TitleElucidation of Attenuation Mechanism of the Ni-CE Strain ofRabies Virus Established from Nishigahara Strain by Passages inChicken Embryo Fibroblast Cells( 本文(Fulltext) )

Author(s) 清水, 健太

Report No.(DoctoralDegree) 博士(獣医学) 甲第227号

Issue Date 2007-03-13

Type 博士論文

Version publisher

URL http://hdl.handle.net/20.500.12099/21410

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Elucidation or Attenuation Mechanism of the Ni-CE

Strain of Rabies Virus Established from Nishigahara

Strain by Passages in Chicken Embryo Fibroblast Cells

(狂犬病ウイルス､鶏腫線維芽細胞継代西ヶ原(NトCE)株の

弱毒化機構の解明)

2006

The United Graduate School of Veterinary Sciences, Gifu University

(Gifu University)

SHIMIZU, Kenta

Elucidation of Attenuation Mechanism of the Ni-CE Strain of

Rabies Virus Established from Nishigahara Strain by

Passages in Chicken Embryo Fibroblast Cells

(狂犬病ウイルス､鶏膝線維芽細胞継代西ヶ原(Ni-CE)株の弱毒化機構の解明)

SHIMIZU, Kenta

CONTENTS

PREFACE

CHAIITER 1

Comparison of the Complete Genome Sequence or Avirulemt Ni-CE Strain of

Rabies Virus with That of the Parental Virulent Nishigahara Strain

Summary

lntroduction

･Materials and Methods

Results

Genomic organization of Ni-CE strain

DifFerences in the noncoding reglOn

Differences in the coding reglOn

Locations of amino acid substitutions in each protein

Discussion

Legends to figures

CHAPTER 2

Involvement of Nucleoprotein, Phosphoprotein and Matrix Protein Genes of

Rabies Virus in Virulence for Adult Mice

S ummary

lntroduction

Materials and Methods

6

7

8

10

12

12

12

13

14

17

24

25

26

28

Results

Recovery of rNi-CE strain from cloned CDNA

Growth in NA cells and pathogenlClty for mice of rNi-CE strain

Growth of chimeric viruses in cultured cells

PathogenlClty Of chimeric viruses in mice

Discussion

Legends to figures

CHAIITER 3

Sensitivity of Rabies Virus to Type I Interferon Is Determined by the

Phosphoprotein Gene: Implications for Viral Pathogenicity

Summary

lntroduction

Materials and Methods

Results

Growth of Ni-CE and Ni strains in IFN-treated NA cells

Growth of CE(Nip) strain in IFN-treated NA cells

ISRE activities in NA cells infected with Ni, NトCE and CE(Nip) strains

ISRE activities in NA cells expresslng the P protein of Ni and Ni-CE strains

DiscⅦssion

Legends to figures

CONCLUSIONS

ACKNOWLEDGMENTS

31

31

32

32

35

38

47

48

49

51

53

53

54

54

56

59

REFERENCES 67

PREFACE

Rabies is a fatal neurological disease that affects all mammals, including humans (42)･

The causative agent, rabies virus, is excreted in saliva from rabid animals and transmitted

to other animals or humans by bites. Followlng a long lnCubation period of two weeks to

three months (occasionally up to six years) (53), patientsdevelop severe neurological

symptoms, such as seizure, paralysIS and coma･ Once the symptoms appear, the patients

almost inevitably die. Desplte the fact that rabies is a vaccine-preventable disease and the

fact that more than loo years have passed since the establishment of the first rabies vaccine

by Pasteur, this disease is distributed widely around the Ⅵ℃rld except for a few countries,

including Japan, United Kingdom, Australia and Sweden. It is estimated that more than

55,000 people die of rabies every year (30). In developing countries in Asia, Africa and

South America, rabies is a serious public health problem: a survey of rabies for the year

1996 (63) showed that more than 99% of human deaths from rabies occurred in developing

countries.

Inactivated rabies vaccines are currently the most popular vaccines being usedfor

prevention of rabies in both humans and animals. However, inactivated vaccines,

especially vaccines derived from tissue culture, are too expensive for vaccination of people

and animals in developlng countries. The high production cost of the vaccine is mainly due

to the requlrement Of large amounts of viral antlgen tO Sufficiently induce a protective

immune response in the inoculated animal. On the other hand, inactivated vaccines from

nerve tissues of rabies virus-infected animals can be produced at a lower cost･ However,

such vaccines are less effective than vaccines derived from tissue culture and can cause

serious side effects, including autoimmune encephalomyelitis, in inoculated animals (27,

57). Inactivated vaccines also require a needle-tipped syringe for delivery, hindering

l

vaccination in developlng COuntries, where a shortage of syrlngeS and needles has

continuously been a serious problem･ The facts described aboveare the main reasons for

the persistence of rabies in many developlng COuntries.

Attenuated live vaccines efficiently elicit a protective immune response with a smaller

amount of the virus, because the vaccine virus propagates and produces viral antlgen in the

inoculated animal. Because of this property, attenuated live vaccinescan generally be

produced at a lower cost than inactivated vaccines. Furthermore, these vaccines can be

delivered by needle-free methods such as oral inoculation (2, 50, 62). Some countries have

successfully reduced the incidence of rabies in wild animals by oral vaccination uslng

attenuated live vaccines (8).However, attenuated live vaccines have a serious problem in

safety. It was reported that the vaccine sometimes cause rabies in inoculated animals (15,

61) due to its residual virulence or pathogenic mutation during viral propagation in the

body. This problem in the safety of attenuated live vaccines is the major Obstacle to the

practical use of the vaccine.

These problems of currently available rabies vaccines indicate the need for the

development of attenuated live vaccines that are safe for the prevention of rabies,

especially ln developlng COunties. For this purpose, it is necessary to elucidate the

mechanism by which rabies virus can be attenuated. However, the molecular mechanism

has not been fully clarified yet.

Rabies virus belongs to the family Rhabdoviridae, genus Lyssavirus･ The genome is

approximately 12,000 bases of nonsegmented negative-sense RNA, encoding five

structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M),

glycoprotein (G) and large protein (L).N, P and L proteins constitute a ribonucleoprotein

(RNP) complex together with viral genomic RNA･ The N protein enwraps viral RNA to

2

form a functional template for transcrlptlOn and replication, while P and L proteins

compose RNA-dependent RNA polymerase. The M protein is located on the inner surface

of the envelope and is involved in assembly and budding of the progeny virion･ The G

protein is anchored to the envelope and partlClpateS in receptor binding, membrane fusion,

and induction of virus-neutralizing antibodies (64).

The rabies virus can be divided into street and fixed viruses. The street virus canbe

rephrased as a field strain of rabies virus･ The fixed virus was first established by Pasteur

after numerous passages of a street virus in brains of rabbits or other animals (7)･During

the passages, the fixed virus slgnificantly loses peripheral infectivlty･ The attenuated

phenotype of the fixed virus enables us to utilize the viruses for fundamental studies and

vaccine production.

The fixed viruses are further classified into virulent and avirulent strains by

pathogenicity for mice: virulent strains kill adult mice after intracerebral (i･c･)inoculation,

whereas avirulent strains do not. Some groups have reported that G protein is a major

determinant for pathogenlClty Of a fixed virus for adult mice: strains that have arglnine or

lyslne at position 333 in the G protein are virulent, while mutants with other amino acids at

this site are avirulent (13, 51, 58). It has also been reported that some biological properties,

such as celトto-cell spread (12),membrane fusion (41) and apoptosis-inducing activity (40,

46), are different between virulent and avirulent strains.

The RC-HL strain is an avirulent strain that is used for the production of inactivated

rabies vaccine for animals in Japan･ The RC-HL strain was established from the virulent

Nishigahara (Ni) strain, which has been maintained by rabbit brain passages, after 294

passages in chicken embryos, 8 passages in chicken embryo fibroblast (CEF) cells, 5

passages in Vero cells and 23 passages in hamster lung (HmLu) cells (22)･ Ito et al･ (24)

3

showed by generatinga chimeric R(G) strain that possesses the G gene of Ni strain in the

background of the RC-HL genome that the G gene of Ni strainis associated with virulence

for adult mice. Furthermore, Takayama-Ito et al. (54, 55) have revealed that amino acids at

position 242, 255, and 268 of the G protein of Ni strain are responsible for viral

pathogeniclty･ Thus, many studies have shown the importance of the G gene in viral

pathogeniclty.

On the other hand, it is also clear that the viral pathogeniclty lS not determined only by

the G gene. In the R(G) strain mentioned above, the pathogenicity is not fully restored to a

level comparable to that of the parental Ni strain. In addition, Yamada et all (66) reported

that chimeric viruses with a slngle gene from the RC-HL strain in the background of the Ni

genome were not attenuated but that viruses with a combination of the G gene and at least

one other gene were attenuated･ However, the mechanism has not been elucidated, mainly

because of the large number of nucleotide substitutions between RC-HL and Ni strains･

The Ni-CE strain is another avirulent fixed virus, which is derived from the Ni strain.

As described above, the avirulent RC-HL strain also orlglnateS from the Ni strain, but their

passage histories are very different･ RC-HL strain was established by a total of 330

passages in various cultured cells･ In contrast, Ni-CE strain was established after 100

passages of Ni strain only in CEF cells (unpublished data)･Hence, the author speculates

that this simple passage history of Ni-CE strain has resulted in a smaller number of

nucleotide substitutions in the genome than the number in the genome of RC-HL strain and,

therefore, that Ni-CE and Ni strains are useful for elucidating the attenuation mechanism of

rabies virus.

In this context, the author tried to determined genetic differences between Ni-CE and Ni

strains･ As described in chapter 1, sequence analysts revealed that NトCE strainis

4

genetically more conservative than RC-HL strain when compared to the parental Ni strain･

Furthermore, the results showed the possibility that the attenuation mechanism of Ni-CE

strain is different from that of RC-HL strain.Consequently, as described in chapter 2, the

author tried to identify viral gene(s) relatedto the difference in pathogenicity between

Ni-CE and Ni strains by generatlng Chimeric viruses with respective genes of Ni strain in

the background of Ni-CE genome･ The results clearly demonstrated that the N, P and M

genes are related to the difference in pathogenicity between Ni-CE and Ni strains･ It has

been reported that P protein of rabies virus inhibits the type l interferon (IFN) signaling

pathway (10, 59). However, the relationship between P protein function and viral

pathogenlClty remains to be elucidated･ Hence, as described in chapter 3, the author tried to

examine whether the P proteins of virulent and avirulent strains inhibit the IFN response･

The results suggest that virulent Ni strain and chimeric CE(Nip) strain that possesses the P

gene of Ni strain in the background of Ni-CE genome acqulre higher resistance to IFN than

that of the avirulent Ni-CE strain through inhibition of the IFN signaling pathwayby the P

protein･ These findings provide useful information for the development ofimproved live

VaCClneS.

5

CIIAPTER 1

Comparison of the Complete Genome Sequence of Avirulent Ni-CE

Strain of Rabies Virus Ⅵ7ithThat or the Parental Virulent

Nishigahara Strain

6

Summary

Rabies virus Ni-CE strain causes nonlethalinfection in adult mice after i.c. inoculation,

whereas the parental Ni strain kills mice･ To clarify the geneticdifferences between Ni-CE

and Ni strains, the author determined the complete genome sequence of Ni-CE strain and

compared it with that of Ni strain that has been previously reported･ The genome ofNi-CE

strain was found to be composed of 1 1,926 bases, which is exactly the same size as that of

Ni strain･ The sizes of all co°ing and noncoding reglOnS Of Ni-CE strain wereidentical to

those of Ni strain, indicatlng that the two strains have the same genomic organization.The

nucleotide substitution rate in the whole genome between Ni-CE and Ni strains wasO･23%,

which was markedly loⅥ′erthan that (1.07%) between Ni strain and avirulent RC-HL strain,

which is also derived from Ni strain, implying that the attenuation mechanism ofNトCE

strain is simpler than that of RC-HL strain･ A previous studydemonstrated that RC-HL and

Ni strains have the lowest sequence homologyln the G gene among five viral genes･ In

contrast, the author showed that the P gene has the highest substitution rate atboth

nucleotide and deduced amino acid levels between Ni-CE and Ni strains.Furthermore, the

amino acids at positions 242, 255 and 268 in the G protein related to thedifference in

pathogeniclty between Ni and RC-HL strains were all conservedbetween Ni-CE and Ni

strains･ These findings suggest that the attenuation mechanism of Ni-CE strainis different

from that of RC-HL strain.

7

Introduction

The genome of rabies virusis a nonsegmented negative-sense RNA composed of

approximately 12,000 nucleotides･ It encodesfive structural proteins: N, P, M, G and L

proteins･ The N, P and L proteinsform a RNP complex with viral genomic RNA･ The N

protein is responsible for encapsidation of the viral genomic RNA, and the L protein,in

cooperation with the P protein, functions as an RNA-dependent RNA polymerase that

synthesizes the viral genome and viral mRNA･ On the other hand, the M and G proteins

form the viral envelope together with a lipid membrane derivedfrom host cells･ The M

protein partlCIPateS in assembly and budding of the progeny virus･The G protein is

specifically responsible for binding to receptors on host cells and theinduction of virus

neutralizing antibody (64)･

The fixed rabies viruses are classified into virulent and avirulent strainsby

pathogenlClty for mice: virulent strains kill adult mice afteri･c･ inoculation, whereas

avirulent strains do not. An avirulent fixed virus, RCIHL strain, was established after

numerous passages of virulent Ni strain in a variety of cultured cells･Ito et al･ (23)

determined and compared fulト1ength genome sequences of RC-HL and Ni strains･The

results showed that the two strains share 98･9% homology ln the nucleotide sequence of

the whole genome, the homology of the G gene being the lowest among five viral genes･

Ito et al. (24) also demonstrated by generating a virulent chimeric virus with the G gene

from Ni strain in the RCIHL genome that theG gene is related to the difference in

pathogenicity between the two strains. In addition,Takayama-Ito et all (54, 55) reported

that amino acids at positions 242, 255 and 268 in the G protein are responsiblefor the viral

pathogenicity. These findings support the results of previous studies (13, 51, 58) showing

that the G protein is a major determinant of the pathogeniclty Of rabies virus･On the other

8

hand, it has been suggested that a viral gene other than the G gene is also involved in viral

pathogenicity. Yamada et al･ (66) generated a chimeric virus with the G gene of RC-HL

strain in the background of the Ni genome and showed that the chimeric virus was not

attenuated, causlng lethal infection to adult mice after i･c･ inoculation･ Furthermore, it was

shown that chimeric viruses with the G gene and at least one other genefrom RC-HL strain

were attenuated, indicatlng that attenuation from Ni strain to RC-HL strain is multigenic･

However, the mechanisms by which other viral genes contribute to the attenuation of

RC-HL strain remain to be elucidated, mainly due to the large number of nucleotide

substitutions between the genomes of RC-HL and Ni strains･

The Ni-CE strain is another avirulent fixed virus, which is also derived from virulent

Ni strain･ It is noteworthy that the passage history of Ni-CE strain is very different from

that of RC-HL strain: In contrast to RC-HL strain obtained after as many as 330 passages

of Ni strain in various cultured cells, Ni-CE strain was established after 100 passages of Ni

strain only in CEF cells･ Thus, it is speculated that this simple passage history of Ni-CE

strain has resulted in a smaller number of nucleotide substitutions in the genome than the

number in the genome of RC-HL strain･ If so, comparison between Ni-CE andNi strains

could enable us to more easily understand the attenuation mechanism of rabies virus･

Hence, to clarify genetic differences between Ni-CE and Ni strains, the author

determined the complete genome sequence of the Ni-CE strain and compared it with that

of the Ni strain that had been previously reported (23). In addition, the genetic differences

between Ni-CE and Ni strains were compared with those between RC-HL and Ni strains･

The author demonstrated that the Ni-CE strain was genetically more conserved than the

RC-HL strain･ The author also showed the possibility that the attenuation mechanism of

Ni-CE strain is different from that of RCIHL strain.

9

Materials and Methods

Cell and virus

Mouse neuroblastoma NA cells were maintained in Eagle's minimum essential

medium (MEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with lO%

fetal calf serum (FCS) (Sigma-Aldrich Corp., St. Louis, MO, USA)･ Ni-CE strain was

established after loo passages of Ni strain in CEF cells (unpublished data)･Virus stock of

Ni-CE strain was prepared in NA cells after clonlng With limltlng dilution three times･ ln

advance of the sequence analysis, the author confirmed that the cloned Ni-CE straindid

not kill adult mice after i.c.inoculation (data not shown).

Reverse transcription (RT), polymerase chain reaction (PCR) and sequence analysis

Total RNA was extracted from the virus stock of NトCE strain using ISOGEN (Nippon

Gene, Tokyo, Japan). Subsequently, single-stranded complementary DNA (CDNA) was

synthesized with Ready-To-Go You-Prime First-Strand Beads (GE Healthcare

Bio-Sciences Corp., Piscataway, NJ, USA). A total of 1 1 CDNA fragments covering most

of the genomic region were amplified by PCR using TaKaRa Ex-Taq (TaKaRa Bio lnc･,

Shiga, Japan) (Fig. I-1).cDNAs of the 3. and 51 terminal regions of the Ni-CE genome

were amplified by rapid amplification of CDNA ends as reported by Ito et al･ (23)･Briefly,

genomic and antlgenOmic RNAs were extracted from NA cells infected with Ni-CE strain

and were added to the adaptor (5'GTA GGA ATT CGG GTT GTA GGG AGG TCG ACA

TTA C 3-) in the respective 3'end using T4 RNA Ligase (TaKaRa I∋io lnc･)･After

purification of the RNA with MicroSpin S-400 HR columns (GE Healthcare Bio-Sciences

Corp.), RT-PCR was performed as described above･ The primers used are listed in Table

1-1.

10

Amplified CDNA fragments were cloned into pT7Blue T-vector (Merck KGaA,

Darmstadt, Germany), and sequencing was carried out with a Thermo Sequenase Primer

Cycle Sequencing Kit (GE Healthcare Bio-Sciences Corp･) and ALF DNA sequencer (GE

Healthcare Bio-Sciences Corp.). To eliminate the influence of misreading of DNA

polymerase, at least three clones were sequenced･ The complete genome sequence of

Ni-CE strain determined in this study was reglStered in DDBJ/EMBL/GenBank database

(Accession no. AB128149).

ll

Results

Genomic organization of Ni-CE strain

The genome of Ni-CE strain was found to be composed of ll,926 bases (Fig･ I-2),

exactly the same size as that of Ni strain (23)･ Five viral genes, N, P, M, G and L genes,

were located on the genome in that order from the 3- terminus･ The lengths of open reading

frames (ORFs) of N, P, M, G and L genes were 1,353, 894, 609, I,575 and 6,384 bases,

respectively, which are also the same as those of Ni strain･ In addition, there wereno

differences in the number of nucleotides in any of the noncoding reglOnS･ Thus, the

genomic organization of Ni-CE strain is identical with that of Ni strain･

Differences in the noncoding region

A total of 28 nucleotide substitutions were found in the whole genome between Ni-CE

and Ni strains, of which three were located in the noncoding reglOn: One Was in the G-L

noncoding region (nucleotide number 4,908) and two were in the 5′ terminal noncoding

region (nucleotide numbers ll,836 and ll,914). The positions and sequences of

transcriptional start and stop signals (23) of respective genes were completely conserved

(Table 1-2).In addition, ll nucleotide complementary sequences in the 3'terminus and 5'

terminus, which are thought to be important for transcription and replication (6),were also

conserved.

●

Differences in the coding reglOn

The substitution rates of the respective co°ing reglOnS between Ni-CE andNi strains

are shown in Tableト3. The nucleotide substitution rate of the P gene (1.12%) was higher

than the nucleotide substitution rates of other viral genes (ranging from O･09% to O･33%)･

12

Similarly, the amino acid substitution rate of P protein (1･68%) was also higher than those

of other viral proteins (ranging from O･14% to O･99%)･

Next, we compared the substitution rates between Ni-CE and Ni strains with those

between RC-HL and Ni strains that had been previously reported (23) (Tableト3)･ The

nucleotide substitution rate in the whole genome between NトCE and Ni strains (0･23%)

was clearly lower than that between RC-HL and Ni strains (1･07%)･ In addition, the

substitution rates in respective co°ing reglOnS between Ni-CE and Ni strains were alllower

than those between RC-HL and Ni strains at both nucleotide and amino acid levels, except

for the nucleotide substitution rate of the P gene･ Notably, the amino acid substitution rate

of G protein between NトCE and Ni strains (0.38%) was markedly lower than that between

RC-HL and Ni strains (2.67%).

Locations or amino acid substitutions in each protein

The locations of amino acid substitutions in N, P, M, G and L proteinsbetween Ni-CE

and Ni strains are shown in Fig･ 1-3･ A total of 15 amino acid substitutions werefound in

various reglOnS･ In the N protein, three amino acid substitutions werefound in the

carboxy-terminal half･ In the P protein, there were five amino acid substitutions, of which

four were in the reglOn ranglng from positions 56 to 81･ In addition, amino acids of these

four positions were all changed to proline, which generally affects the protein structure･ ln

the M and G proteins, two amino acid substitutions were found in the amino-terminal half,

respectively･ The L protein had three amino acid substitutions, two locatedin the

amino-terminal reglOn and one in the central reglOn･ The amino acids at positions242, 255,

and 268 in the G protein, which were shown to be related to thedifference in pathogeniclty

between RC-HL and Ni strains (55),were all conserved between Ni-CE and Ni strains･

13

Discussiom

To clarify the geneticdifferences between the avirulent NトCE strain and the parental

virulent Ni strain, the author determined the complete genome sequence ofNi-CE strain

and compared it with that of Ni strain that has been previously reported (23)･As a result, a

number of genetic differences between the two strains became apparent･ It shouldbe noted

that amino acids at positions 242, 255 and 268 in the G protein, which were identifiedas

determinants of the difference in pathogenicity between Ni and RCIHL strains (55), were

all conserved between Ni-CE and Ni strains. Furthermore, the amino acid at position333

in the G protein, a well-known determinant of pathogenicity of rabies virus (13, 51, 58),

was also conserved･ These results suggest that the attenuation mechanism ofNi-CE strain

is different from that of RC-HL strain as well as many other strains･

The Ni-CE strain was genetically more conserved thanRC-HL strain when compared

to the parental Ni strain, especially in the G gene, whichis important for entry of the virus

into host cells･ This difference is probably due to passagehistories of these strains: Ni-CE

strain Ⅵ′as established from Ni strain by 100 passages onlyin CEF cells, while RC-HL

strain was established by a total of 330 passages in a variety of cultured cells･

Many studies have shown that G protein of rabies virus is closely associated with viral

pathogenicity (13, 24, 51, 54, 55, 58). As shown in Fig･ I-3, there were two amino acid

differences in the G protein between Ni and Ni-CE strains (atpositions 50 and 182)･ They

were both radical amino acid substitutions that may alter the conformation of the protein･

The amino acid substitution at position 182 was adjacentto the putative binding domain

(residues 189 to 214) for nicotinic acetylcholine receptor (nAChR), which is thought to be

one of the receptors of rabies virus (33, 34). The amino acid substitution at position 50 was

located around the putative N-linked glycosylation site (at position 37) (52)･ This

14

glycosylation site is probably ln prOXimlty tO the binding domain for nAChR in the steric

structure, because both domains Ⅵ′ere included in the antlgenic site II comprised of the

discrete regions in the G protein (residues 34 to 42 and 198 to 200) (45).Therefore, these

amino acid substitutions may influence the glycosylation efficiency and the affinity of the

virus for nAChR.

Alternatively, it is possible that viral proteins other than G protein are related to

pathogenlClty･ Among five viral proteins, P protein showed the highest amino acid

substitution rate (1.68%) (Tableト3). In addition, amino acids at positions 56, 58, 66 and

81 in the P protein were all changed to proline, which generally affects the protein

structure (Fig. l13),suggesting that the P protein structure of Ni-CE strain was different

from that of Ni strain･ Furthermore, some amino acid substitutions in the P protein were

located in or around the functional motifs that are required for the nucleocytoplasmic

transport of P protein (43). The amino acid substitutions at positions 56 and 58 were

located in the nuclear export signal (residues 49 to 58). The amino acid substitution at

position 226 was in proximity to the nuclear localization signal (residues 211 to 214 and

260), as shown in the crystal structure of P protein (35). Therefore, the distribution of P

protein in infected cells may be altered by these amino acid substitutions.

Besides the P protein, N and L proteins, other components of the RNP complex, also

contained some amino acid substitutions around functional domains. The amino acid

substitution at position 273 in the N protein was located in the central reglOn, Which is

involved in binding of N protein to the viral RNA (1, 31). In addition, the amino acid

substitutions at positions 394 and 395 in the N protein were included in the

carboxy-terminal region, which is needed for binding of N protein to the P protein (49).

The radical substitution at position 1,079 in the L protein was close to the conserved block

15

IV (residues 889 to 1,060) and block V (residues 1,090 to 1,326) present in the RNA

polymerase of the mononegavirales group of negative-strand RNA viruses (44), which is

thought to be important for the enzymatic activlty･ Thus, these amino acid substitutions

may innuence the function of the RNP complex for transcrlptlOn and replication of the

viral genome.

Amino acid substitutions in the M protein were located at positions 29 and 95, which

were highly conserved among members of the genus Lyssavirus, thus implying the

functional importance of the amino acids of these positions･ The amino acid substitution at

position 29 was close to the PPXY motif (residues 35 to 38), which is involved in the

budding process through binding to the WW domain of host proteins (18, 19)･The amino

acid substitution at position 95 Ⅵ′as located in a highly hydrophobic region (residues 89 to

107) that is thought to be associated with the host membrane (56)･ The assembly and

budding processes may therefore be affected by these amino acid substitutions･

The noncoding reglOn Of the viral genome contains slgnal sequences that are important

for transcrlPt10n and replication･ In some viruses, nucleotide substitutions in the noncoding

region also lead to alteration of viral pathogenicity (11)･Although the transcriptional start

and stop signals (23) and the terminal complementary sequences (6) were conserved

betⅥ′een NトCE and Ni strains, the substitution at the genome nucleotide number ll,914

was very close to the 5'terminal complementary sequence (nucleotide number ll,916 to

1 1,926).Therefore, this substitution may impair some functions relating to the sequence･

In conclusion, the author has clarified the difference of genetic backgroundbetween

Ni-CE and Ni strains, and the author has also shown the possibility that Ni-CE strain is

attenuated by a novel mechanism.

16

Legends to figures

Fig･ 111･ Schematic diagram of genomic positions of amplified CDNA fragments (a-m).

Details of annealing positions and sequences of respective prlmerS are Shown in Table 1-1.

Fig･ 1-2･ Genomic organization of Ni-CE strain･ Squares represent ORFs of each gene･

Numbers indicate the number of nucleotides of respective reglOnS･ Numbers in parenthesis

indicate the number of deduced amino acids of each protein･

Fig･ 1-3･ Amino acid substitution sites in N, P, M, G and L proteins of Ni-CE strain

compared to the parental Ni strain･ Asterisks indicate the amino acid changes from Ni

strain to NトCE strain･ Numbers represent the changed amino acid positions･ The amino

acid number in G protein is asslgned to the mature form that does not contain a slgnal

peptide (hatched).

17

Table 1-I

Primers used for sequence analysis

Fragment*1 Name Sense Position*2 sequence (5'to 3T)

RHN-1 +

RHNS-3

RHN-6 +

RHN-7

RUNS-1 +

RGP-13

Pl +

RGPst I

RGP-14 +

RHL-27

RHL-26 +

RHL-7

RHL-6 +

RHL-9

RHL-8 +

RHL-23

RⅢL-12 +

RHL-13

RHL- 24 +

RⅢL-1 5

RHL-25 +

R日L-17

SSON-2 +

RHN-2

RHL- 19 +

SSON-2

28-50

1517-1539

1415-1435

2482-2502

2386-2405

3386-3405

3290-3313

491ト4930

4752-4775

5429-5452

5384-5408

6742-676 1

60611608 1

740 1-7420

6715-6735

8274-8293

8058-8077

8962-8982

8790-8809

10501-10521

10245- 10264

1188ト11901

SSON adaptor

562-584

11372-11391

SSON adaptor

ACA GA〔 AGC GTCAAT TGC AAA GC

TCG GATTGA CGAAGATCTTGC TC

GA〔 TCT TAA GGA GTT AAA CAA

GTT CAT TTT ATC ACT GGT GTT

TGA ATC GCT ATG CAT CTT GC

AGTGTG TCT GGT ATC GTG TA

AÅc ATC CCT CAAAAG ACTTAA GGA

CAT CTG CAG AÅc TTGAAG CG

GTT GTA GAAAAGTCG ATC GGC CAG

GGATCA ATG GGG TCATCA TAG ACC

ACCTCTAAG CTT GAAACC TAC ATC

TAC AAT ATG TTT GGG TGG CC

AAA TAT GGG GAC TGC TTA TTG

CCA ATG AGG TCT GAT CTG TC

TGA CTC CTT ATG TCA AAA CCC

AGAAGG CGA GTG AAG CTCTC

AGC TTT CTC CTA GCT ATG TC

TCTTCA CCA CAT GAA CAT TAG

AAG TCCTCT ATTTCTTGCAC

CAA CTA CAA GGC AGA GAG ATG

GCA GCTAAAACC ATG ACT GG

TGA GAA AAA CAA TCA AÅc AÅc

TCC CTA CAA CCC GAATTC CT

TCT GCT CTA TCC TAT CTG CAA TG

TGA CCC CAT GTT CTA TCCAC

TCC CTA CAA CCC GAATTC CT

*1The fragments are shown in Fig･ 1-1･

*2The numbers are based on the genome nucleotide number of Ni strain.

18

Table 1-2

TranscrlptlOnal start and stop slgnals of NトCE strain

Gene Start Position* Stop Position

N

P

M

G

L

AACACCTCT

AACACCTCT

AACACCACT

AACAT CCCT

AACACCTCT

59-67

1485-1493

248 2- 2490

329013298

5382-5390

TGAAAAAAA 1474- 1482

T GAAAAAAA 2468-2476

TGAAAAAAA 3276-3284

AGAAAAAAA 5349-5357

CGAAAAAAA 1 1848-1 1856

*The numbers are based on the genome nucleotide number of Ni-CE strain.

19

Tableト3

Substitution rates (%) of Ni-CE and RC-HL strains compared to the parental Ni strain

Strain Sequence

N P M G L

Genome

Ni-CE/Ni*1 nt*2

aa*3

RC-HL*4/Ni nt

aa

0.22 1.12

0.67 1.68

0.81 1.12

1.11 2.02

0.33 0.25

0.99 0.38

0.99 1.46

1.49 2.67

0.09 0.23

0.14

0.85 1.07

0.85

*1GenBank accession no. ABO44824.

*2Nucleotide.

*3Amino acid.

*4GenBank accession no. AI∋009663.

20

g+

h-

1 +

u二二

e +

f+

Fig. 1･1

21

3ー

11926^L

5ー

1r

l35389460915756384^L

(297)

P

(202)

M

.JL ..一L1r

(450)1r

(524)山llllll■■■■一山

(2127)

N G L

70 91 88 211 520 131

Fig. 1･2

22

☆ ☆ 450 aa

273 : F･･.うL 394: Y.i'H

395 : F→L

倣 ☆ 297 aa

56,58,66 : L-P 226: N-H

81:F→P

M ☆ ☆29: D→E

95:Ⅴ→A

202 aa

☆ ☆

50:Y→R 182:S→Ⅰ

100 aa: for N, P, M, and G proteins

505 aa

61 : C･･･⇒R

121 :A→V

100 aa: for L protein

■■■■■■

1079 : R-Q

Fig. 1-3

23

2127 aa

CHAPTER 2

Involvement or Nucleoprotein, Phosphoprotein and Matrix Protein

Genes of Rabies Virus in Virulence for Adult Mice

24

Summary

To identify viral gene(s) related to the difference in pathogenicity between Ni-CE and

Ni strains, the author generated chimeric viruses with respective genes of the virulent Ni

strain in the background of the avirulent Ni-CE genome･ Since chimeric viruses that had

the N, P or M genes of the Ni strain, respectively, killed adult mice after intracerebral

inoculation, it became evident that the N, P and M genes are related to the difference in

pathogeniclty between Ni-CE and Ni strains･ Previously, we shoⅥ7ed that the G gene is a

major contributor to the difference in pathogeniclty between Ni strain and avirulent RCIHL

strain, which is also derived丘･om Ni strain･ These results provide evidence that the

attenuation mechanism of NトCE strain is different from that of RC-HL strain, thus

suggestlng that rabies virus can be attenuated by diverse mechanisms. This is the first

report of changes in viral genes other than the G gene of rabies virus causlng the reversion

of pathogeniclty Of an avirulent strain.

25

Introduction

Many studies have shown the importance of the G gene in rabies virus pathogeniclty･

The amino acid at position 333 of the G protein is a welトknown determinant of

pathogenicity (13, 51, 58). Takayama-Ito et al･ (54, 55) have also shown that multiple

amino acids at positions 242, 255 and 268 of the G proteinare related to the difference in

pathogeniclty between RC-HL and Ni strains･ As shown in chapter 1, the author clarified

genetic differences between the avirulent Ni-CE strain and the parental virulent Ni strain･

Notably, amino acids at positions 242, 255 and 268 as well as at position 333in the G

protein were all conserved between Ni-CE and Ni strains･ The results suggest that the

attenuation mechanism of Ni-CE strain was different from the attenuation mechanisms of

RC-HL strain and many other strains.

Between Ni-CE and Ni strains, the amino acid substitution rate of P protein was the

highest among five viral proteins･ In addition, the P protein contained a cluster offour

amino acid substitutions, all of which were changed to proline residue, whichis thought to

affect the protein structure･ On the other hand, the amino acid substitution rate ofG protein

was the second-lowest: only two amino acid substitutions were found･ These results raise

the possibility that viral genes other than the G gene are involved in viral pathogeniclty･ In

order to identify the viral gene related to the difference in pathogenlClty between Ni-CE

and Ni strains, the author sought to generate a series of chimeric viruses between the two

strains with respective genes from Ni strain in the background of the Ni-CE genome, and

to examine whether the chimeric viruses kill adult mice after i.c. inoculation. For this

purpose, it was necessary to manlpulate the genome of Ni-CE strain uslng a reverse

genetics system, which is known as a method to recover a recombinant virus from cloned

CDNA.

26

In this chapter, the establishment of a reverse genetics system of Ni-CE strain and

generation of a series of chimeric viruses between Ni-CE and Ni strains are descrived.

Examination of the pathogeniclty Of the chimeric viruses for adult mice made showed that

the N, P and M genes are involved in the attenuation of Ni-CE strain.

27

Materials and Methods

Cells andviruses

Mouse neuroblastoma NA cells Were maintained in Eagle's MEM supplemented with

lO% FCS. A baby hamster kidney (BHK-21) cell clone, BHK/T7-9 cells (25), which

constitutively express T7 RNA polymerase, were maintained in Eagle's MEM

supplemented with lO% tryptose phosphate broth (Becton, Dickinson and Company,

Franklin Lakes, NJ, USA) and 5% FCS. Recombinant (r)Ni strain was recovered froIⅥ the

cloned CDNA of Ni strain as reported by Yamada et al. (66). Virus stocks of rNi and

NトCE strains were prepared in NA cells.

RTIPCR and sequenclng●

cDNA fragments Ⅵ′ere amplified by RT-PCR uslng prlmerS Shown in Table 2-1 as

described in chapter l･ After cloning of CDNA fragments into pT7Ⅰ】1ue T-vector (Merck

KGaA), sequencing was carried out with a Dual CyDye Terminator Sequencing Kit (GE

Healthcare Bio-Sciences Corp.) and Long-Read Tower (GE Healthcare Bio-Sciences

Corp.).

Construction of full･length genome plasmid

A full-length genomic CDNA of Ni-CE strain was constructed on pUC19 by stepwise

subclonlng uSlng CDNA fragments derived from the genomic RNA of Ni-CE and Ni

strains as reported by Ito et al. (24) (Fig.2-1). A nucleotide change at nucleotide number

ll,914 was introduced by using a U.S.E. mutagenesis kit (GE Healthcare Bio-Sciences

Corp･) with an L-RCE primer shown in Table 2-1. In order to distinguish the rNi-CE strain

recovered from the full-length genome plasmid from wild-type (wt) Ni-CE strain, in

28

addition to the Pstl site used asa genetic marker for the rNi strain, a second genetic marker,

〟7〟I site, was constructed in the G-L noncoding reglOn Of the genome plasmid by

changing two nucleotide residues at positions 4,914 (T to G) and 4,925 (A to C).

Fulト1ength genome plasmids of chimeric viruses were similarly constructed uslng a

conventional technique.

Recovery of recombinant viruses

Recombinant viruses were recovered from the fulト1ength genome plasmids uslng a

reverse genetics system as reported by Ito et al. (25). ln this system, the T7 RNA

polymerase-expresslng VaCCinia virus that causes homologous recombination of plasmid

DNAs (20) was not used. Briefly, three helper plasmids (pT7IRES-RN, -RP, and -RL)that

possessed a T7 promoter and an internal ribosomal entry site upstream of N, P and L genes

from the RC-HL strain were transfected to BHK/T7-9 cells with respective fulト1ength

genome plasmids using TransIT-LTl (Mirus Bio Corp., Madison, WI, USA)･ After

incubation for 5 to 7 days, viruses in culture supernatants were collected･ Stocks of

recombinant viruses were prepared in NA cells. The authenticlty Of each gene of recovered

viruses was confirmed by restriction endonuclease digestion and/or partial sequenclng Of

RT-PCR fragments.

Confirmation of the presence of the genetic marker

Using RGP-14 (5'-GTT GTA GAA AAG TCG ATC GGC CAG13') and RHL-27

(5'-GGA TCA ATG GGG TCA TCA TAG ACC-3') primers annealing at nucleotide

numbers 4,752 to 4,775 and 5,429 to 5,452, respectively, a cDNA什agment was amplified

from the genome of each recombinant virus by RT-PCR･ The amplified product was

29

treated with restriction endonuclease 〟/〟I and electrophoresedon 1.5% agarose gel.

Propagation or recombinant viruses in NA cells

NA cells grown in a 6-well tissue culture plate (Greiner Bio-One Co･ Ltd･, Tokyo,

Japan) were inoculated with each virus at a multiplicity of infection (MOI) of O･01･ At 1, 3

and 5 days post-inoculation (dpi),viruses in the culture supernatants were harvested and

titrated in NA cells by lmmunOfluorescence assay uslng a mOnOClonal antibody 81l

specific for N protein (39)･

Pathogenicity of recombinant viruses in mice

Five 6-week-old ddY female mice (Japan SLC Inc., Shizuoka, Japan) per group were

inoculated by the i.c. routewith 30トLl of 10, 100 and 1,000 focus-forming units (FFU) of

each virus, respectively. The mice were observed daily for neurologlCal symptoms and

classified into four grades: normal, mild neurological symptoms (such as wobble and

motor incoordination), severe neurological symptoms (such as paralysis, seizure, and

coma), and dead. Five2-day-old ddY mice (Japan SLC Inc･)per group were inoculated by

the i.c. route with 15ト1l of serial ten-fold dilutions of rNi-CE and wtNi-CE strains･ The

fifty percent of lethal dose (LD5.) was calculated by the method of Reed and Miiench (47)･

30

Results

Recovery of rNi-CE strain from cloned CDNA

To generate genetically modified viruses with the background of the Ni-CE genome,

the author established an infectious CDNA clone of the Ni-CE strain. To confirm that the

rNトCE strain was derived from the full-length genome plasmid, the author checked the

presence of the genetic marker, 〟/〃I site, 1n the G-L noncoding reglOn by RT-PCR and

restriction endonuclease digestion (Fig. 2-2A). An amplified CDNA fragment from the

recovered virus was cleaved into two fragments with the expected size after treatment with

MluI, whereas that from wtNi-CE strain was not cleaved (Fig. 212B, lanes 2, 3, 5 and 6).

When PCR was performed without the RT step, no fragment was amplified (Fig･ 2-2B,

lanes 1 and 4), indicating that the amplified CDNA fragment did not originate from the

fulト1ength genome plasmid used in the virus recovery process･ Thus, the author

successfully recovered rNi-CE strain from cloned CDNA.

Growth in NA cells and pathogenicity for mice of rNi-CE strain

The author compared the growth characteristic in NA cells and the pathogenlClty for

mice of rNi-CE and wtNi-CE strains. Fig. 2-3A shows the multistep growth curves of each

virus in NA cells. The virus titer of rNi_CE strain in the culturefluid reached log FFU/ml

by 3 dpi, which was comparable to that of wtNi-CE strain･ In addition, the growth curve of

rNi-CE strain was almost the same as that of wtNi-CE strain. Fig. 2-4A shows the body

weight change of adult mice inoculated with 1,000 FFU of each virus by the i･c･ route･ The

body weight of mice inoculated with rNi-CE strain began to decrease from 6 dpi, becaIⅥe

lowest around 10 dpi (about lo啄 reduction),and thenincreased･ The body weight curve of

mice infected with rNi-CE strain was similar to that of mice infected with wtNi-CE strain.

31

During a 2-week observation period, none of the mice developed neurologlCal symptoms

or died of infection. Meanwhile, rNi-CE and wtNi-CE strains caused lethal infection in

suckling mice (1.5 and I.7 FFU of LD5｡, respectively).Thus, growth in NA cells and

pathogenlClty for mice of rNi-CE strain were almost the same as those of wtNi-CE strain.

GroⅥ7th or chimeric viruses in cultured cells

To determine which genes are related to the difference in pathogeniclty between Ni-CE

and Ni strains, the author generated chimeric viruses, CE(NiN), CE(Nip), CE(NiM),

CE(NiG) and CE(NiL) strains, by replacement with respective genes of the virulent Ni

strain in the background of the avirulent Ni-CE genome. The presence of the genetic

marker, Mlul site, in the GIL noncoding reg10n Of each virus was confirmed as described

above (data not shoⅥ′n).In addition, the author determined the partial sequence of the

respective modified genes by direct sequenclng and confirmed that it was identical to the

authentic sequence.

Next, the author examined the growth characteristics of chimeric viruses in NA cells

(Fig. 2-3B). The virus titers of CE(NiN), CE(Nip), CE(NiG) and CE(NiL) strains in the

culture fluid reached about 108 FFU/ml, respectively, which were comparable to those of

rNi-CE and rNi strains. In addition, the growth curves of these viruses were also similar to

those of rNi-CE and rNi strains. On the other hand, the virus titer of CE(NiM) strain in the

culture fluid reached only about 106 FFU/ml, although the titer at 1 dpi was comparable to

others.

Pathogenicity of chimeric viruses in mice

The author evaluated the pathogeniclty Of chimeric viruses by i.c. inoculation of adult

32

mice. When mice were inoculated with 1,000 FFU of viruses, rNi-CE strain did not kill

any mice (Table 2-2).In contrast, CE(NiN), CE(Nip) and CE(NiM) strains, which had the

N, P or M genes of the virulent Ni strain in the background of the avirulent Ni-CE strain,

respectively, killed all mice. On the otherhand, CE(NiG) and CE(NiL) strains killed 20%

and O% of mice, respectively･ Even when mice were inoculated with a higher dose of

cE(NiG) (106 FFU) or CE(NiL) (105 FFU) strain, the mortality rates did not increase (data

not shown). The mortality rates of miceinoculated with 10 FFU of CE(NiN), CE(Nip) and

CE(NiM) strains were 40%, 80% and 60%, respectively, in contrast to the mortality rate of

mice inoculated with rNi strain at the same dose (loo鞄).

The body weight changes in mice inoculated with 1,000 FFU of each virus are shown

in Fig. 2-4B. The mice inoculated Ⅵ′i也rNi-CE and CE(NiL) strains lost body weight

transiently. In contrast, the mice inoculated with CE(NiN), CE(Nip), and CE(NiM) strains

continued to lose body weight, resulting in death, although these mice began to losebody

weight later than those inoculated with rNi strain･ The mice inoculated with CE(NiG)

strain also lost body weight by 9 dpi, as did the mice inoculated with CE(NiN), CE(Nip)

and CE(NiM) strains, but they later recovered.

The morbidity and mortality changes in mice inoculated with 1,000 FFU of each virus

are shown in Fig･ 2-5･ The mice inoculated with rNi-CE strain did not show neurologlCal

symptoms. In contrast, all of the miceinoculated with CE(NiN), CE(Nip) and CE(NiM)

strains developed severe neurologlCal symptoms anddied of disease by 14 dpi, although

the onset of disease was delayed compared to that in the case of rN strain･On the other

hand, CE(NiG) and CE(NiL) strains caused mild neurological disease in mice･ Only one of

the mice inoculated with CE(NiG) strain died at 6 dpi.

The results revealed that CE(NiN), CE(Nip) and CE(NiM) strains had markedly higher

33

pathogenicity for mice than did rNトCE, CE(NiG) and CE(NiL) strains, although these

virulent chimeric strains did not completely regaln pathogeniclty comparable to that of rNi

strain.

34

Discussion

To identify viral gene(s) relatedto the difference in pathogenicity between the avirulent

Ni-CE strain and the parental virulent Ni strain, the author generated chimeric viruses with

respective genes of Ni strain in the background of the Ni-CE genome･Since chimeric

viruses that had the N, P, or M genes of the Ni strain, respectively,killed adult mice after

i.c. inoculation, it became evident that the N, P and M genes are related to thedifference in

pathogenlClty between Ni-CE and Ni strains･

Recently, it has been reported that the P protein of rabies virus counteracts the host

antiviral responses in vitro. Brzozka et all (9) demonstrated that the P protein of rabies

virus prevented type I interferon (IFN) response by interfering with phosphorylation of

IFN regulatory factor 3. In addition, Vidy et all (59) reported that the P protein of rabies

virus interacted with the signal transducer and activator of transcription1 (STATl) and

inhibited the IFN signal transduction pathway by preventlng IFN-induced STAT 1 nuclear

accumulation. Furthermore, Blondel et al. (3) showed that the P protein of rabies virus

interacted with IFN-induced promyelocytic leukaemia (PML) protein and reorganized

PML nuclear bodies, which is thought to be a part of thehost defense mechanism (5, 14)･

Although the slgnificance of these functions of the P proteins of rabies virus in vivois

unclear, the author speculates that these functions a什ect thedifferences in the viral

pathogenicity between rNi-CE and CE(Nip) strains･ It is notable that some of the amino

acid substitutions in the P protein of the Ni-CE strain are present around thefunctional

motif required for the nucleocytoplasmic transport of P protein (43), as described in

chapter 1: amino acid substitutions at positions 56 and58 of the NトCE strain are located in

the nuclear export signal (residues 49 to 58), and the amino acid substitution at position

226 is in proximity to the nuclear localization signal (residues 211 to 214 and 260), as

35

revealed by the crystal structure of P protein (35)･ Therefore, these amino acid

substitutions may change the distribution of P protein in the cytoplasm and nucleoplasm of

infected cells, resulting ln alteration of some viral functions such as the prevention of

STATl nuclear accumulation (10,59) and the reorganization of PML nuclear bodies (3).

The role of nucleoprotein in viral patbogenicltylS poorly understood･ The KKYK motif

on the P protein, a part of the nuclear localization slgnal mentioned above, is also involved

in the binding to the N protein (26).This implies a modulating role of the N protein in the

transport of P protein･ Interestlngly, two amino acid substitutions at positions 394 and 395

of the N protein of Ni-CE strain are included in the carboxy-terminal reglOn needed for

binding to P protein (49). These amino acid substitutions may affect the transport of P

protein in cells and consequently influence the ability of P protein, which can inhibithost

antiviral responses.

When the growth characteristics of viruses in NA cells were compared, CE(NiM) strain

showed lower growth efficiency than that of the others, including rNi-CE and rNi strains

(Fig. 2-3). This suggests that the M gene of the Ni strain may be incompatible with other

components of the Ni-CE genome･ This phenomenon may be attributed to theimpalrment

of M protein functions, such as budding of virus particles (38) and regulation of RNA

synthesis (16), which are important for efficient viral growth･ The amino acid substitution

at position 29 of the M protein of Ni-CE strain was close to the PPXY motif, which is

thought to be involved in the budding process through interaction with WW domains of

cellular proteins (18, 19).In addition, amino acid substitution at position 95 is located in a

highly hydrophobic region (residues89 to loワ)(56),which is thought to be associated with

the host membrane･ Therefore, changes in these amino acids may affect theinteraction

with other molecules and lead to an unbalanced relationship among viral andhost proteins.

36

Interestingly, the CE(NiM) strain caused lethal infection in adult mice after i.c.

inocu)ation in splte Of inefficient growth in NA cells･ There is no report of changes only ln

the M gene of rabies virus altering the viral pathogenicity･ Kassis et al･ (29) demonstrated

that M proteins of Mokola and Lagos bat viruses, members of the genus LJySSaVirus,

induced apoptosis by a tumor necrosis factor-related apoptosis-inducing ligand-dependent

mechanism. In addition, it has been reported that the pathogenlClty Of rabies virus is

inversely correlated with apoptosis (40, 46). Although the role of the M protein of rabies

virus in apoptosis-inducing activlty lS unClear, it is possible that amino acid substitutions of

the M protein may affect this activlty and alter the viral pathogeniclty･

The avirulent NトCE and RC-HL strains were established from the virulent Ni strain by

different passages. Previously, Ito et al. (24) showed that a chimeric R(G) strain with the G

gene of the Ni strain in the background of the RC-HL genome killed adult mice after i･c･

inoculation as did the Ni strain. In contrast, the CE(NiG) strain with the same G gene of

the Ni strain in the background of the Ni-CE genome only caused mild neurologlCal

disease and did not kill most mice. These results provide evidence that the attenuation

mechanism of Ni-CE strain is different from that of RCIHL strain, thus suggesting that

rabies viruses can be attenuated by diverse mechanisms･

In conclusion, the author clearly demonstrated that the N, P and M genes are involved

in the difference in pathogeniclty between Ni-CE and Ni strains. This is the first report of

changes in viral genes other than the G gene of rabies virus causlng reversion of

pathogeniclty Of an avirulent strain.

37

Legends to figures

Fig. 2-1. Construction of full-length genome plasmid of Ni-CE strain. A total of nine

CDNA fragments (a-i)were amplified from the Ni-CE genome by RT-PCR and assembled

by stepwise subcloning. Some CDNA fragments (hatched) were derived from plasmids

used for construction of a full-length genome plasmid of Ni strain (66).Numbers are based

on the genome nucleotide number of Ni-CE strain. The genetic marker, Mlul site,

orlglnated from the prlmer Sequence.

Fig. 2･2. Confirmation of the presence of the genetic marker, Mlul site, in the G-L

noncoding region of the rNi-CE genome. (A) Schematic diagram of G-L noncoding region

of the rNi-CE genome, annealing positions of prlmerS, and predicted sizes of amplified and

digested CDNA fragments. (B) Amplified and digested CDNA fragments･ Using total RNA

extracted from the stock of wtNi-CE or rNi-CE as a template, PCRwas performed with or

without the RT step (lanes 1, 2, 4 and 5).The amplified CDNA fragments were treated with

the restriction endonuclease 〟7〟Ⅰ (1anes 3 and 6). Numbers indicate the length of

respective CDNA fragments. M, Molecular size marker･

Fig. 2･3. Growth curves of each virus in NA cells. (A) Growth of avirulent wtNi-CE strain,

rNi-CE strain that is recovered from cloned CDNA in this study, and virulent rNi strain that

is generated by Yamada et al. (66).(B) Growth of chimeric CE(NiN), CE(Nip), CE(NiM),

CE(NiG) and CE(NiL) strains that possesses respective genes of the virulent Ni strain in

the background of the avirulent NトCE genome･ NA cells were inoculated with each virus

at a MOI of 0.01. The virus in the culture fluid was harvested at 1, 3 and 5 days

post-inoculation and titrated in NA cells･ The genome compositions of each strain are

38

shown in schematic diagrams･ The Ⅵ7hite and grey squares represent the genes that

orlglnate from Ni-CE and Ni strains, respectively･

Fig･ 214･ Changes in body weight of adult mice inoculated intracerebrally with I,000 FFU

of wtNi-CE, rNi-CE, rNi (A) and chimeric viruses (B). For mock infection, mice were

inoculated with a diluent･ The changes are shown as ratios considering body Ⅵ′eights of

mice at day 0 as l･ The valuesin the graph are averages and standard deviations of the

ratio. Asterisks indicate the time polnt at Which all of the mice died of disease･

Fig･ 2･5･ Morbidity and mortality changes in adult mice inoculated intracerebrally with

1,000 FFU of each virus. The mice were observed for neurologlCal symptoms for 14 days･

39

Table 2-1

Primers used for construction offull-genome plasmids

ment*1 NameSense Position*2 sequence (5rto 3')

a

b

C

d

e

f

g

h

1

*4

RHN-27 +

RHN-14

RHN-13 +

RHNS-10

RHNS-6 +

RNM-1

Pl +

RG PstI

PIO +

CEMluI

CEL-1

RNL-2

RHL18

RHL-23

RHL-12

RⅢL-13

RHL-19

GRbz31+5.I

L-RCE

+

+

+

+

+

52-74 AAA AGC TTA CAC CTC TA仁 AAT GG

1421-1∠143 CCG CAT CCT TGT TTA ACT CCT TA

1017-1039 GAT GCCATA TGG GTCAAA TCA GA

203ト2050 GAG GCA GTT TGA GCC ATC GT

1958-1977 CAC TAG TCA AGA GCC CAA GA

3106-3125 ACC CGC GGG ATA CAG TCT GA

3290-3313 AAC ATC CCT CAA AAG ACT TAA GGA

491ト4930 GAT CTG CAG AÅc TTG AAG CG

4005-4027 ATG GAA AGA GGC CTA TAT AAG TCT

4906-4929 ATC TGC AGA ACT TGA CGC GTC TAA

*3

4918-4941 AGT TCT GCA CAT CAC CTT CCC TCT

5913-5936 CAA AGA CGT CGA ATA CGT GTT GGC

6715-6735 TGA CTC CTT ATG TCA AAA CCC

8274-8293 AGA AGG CGA GTG AAG CTC TC

8058-8077 AGC TTT CTC CTA GCT ATG TC

896218982 TCT TCA CCA CAT GAA CAT TAG

11372-11391 TGA CCC CAT GTT CTA TCCAC

l1916111926 GAA GAC CGA CCCACG CTT AACAA

I 1901-11926 GTC TTT GTT GTT TGT TTG TTA AGC

GTG GG

*1The fragments are indicated in Fig･ 2-1･

*ユThe numbers are based on the genome nucleotide number of NトCE strain･

*3〟7〟I site for a genetic marker･

*4rhe primer was used for mutagenesis･

40

Table 2-2

Mortality rates (%) in adult mice intracerebrally inoculated with each virus

inoculation dose (FFU/mouse)

1000 100 10

rNi

rNi-CE

CE(NiN)

CE(Nip)

CE(NiM)

CE(NiG)

CE(NiL)

100

0

100

100

100

20

0

41

Genomic RNA

blO171享 リ2050

Full-len-gth genome plasmid

4005

Mlu I

【±ヨ4929d圭

4

h8058 " 8982

蛋

Bsp1407I BstXI … NsiI

1

1 1372 【=コ1192(～

BglII KpnI

AatII

Fig. 2･1

42

A

RT-PCR

〟J〟I digest

Mlu I

-IRGP- 14

⊂=

RH L-27

RT - + +

PCR + + +

Mlul - - +

wtNj-CE rNi-CE

Fig. 2-2

43

】三!1'.㌔

■158

A

育Ii己!ヨ

=)至至LL

bDO

,:

ヽ_′′

ヨ≡

q)

･≡〔/〕

コ

.i:>

9

6

3

0

△ wtNi-CE

orNトCE

■ rNi

3 5 1 3 5

Days post-inoculation

N P M G L

◆ CE(NiN)

▲ CE(Nip)

● CE(NiM)

□ CE(NiG)

◇ CE(NiL)

Fig. 2-3

44

N P M G L

A

C)

'=cd己≡

E=コ

,z=

resa弓ロー■⊂)C)

〔□

B

0

'=cdtZ

+J

..;=

･5iieZl

｢⊃○

〔ロ

1.0

0.8

0.6

0.4

H=3:

0.8

0.6

0.4

7

Days post-inoculation

7

Days post-inoculation

Fig. 2-4

45

× mock

△ wtNi-CE

o rNトCE

■rNi

◆ CE(NiN)

▲ CE(Nip)

● CE(NiM)

□ CE(NiG)

◇ CE(NiL)

q)

U

'fOi2

q)

,.⊂)≡コ

Z

5

4

3

2

1

5

4

3

2

1

0

5

4

3

2

1

0

5

4

3

2

1

0

Fig. 2-5

46

7 14

Days post-inoculation

CE(NiN)

5

4

3

2

1

0

5

4

3

2

1

0

5

4

3

2

1

0

CE(NiG)

□ Normal

[コMildneurological symptoms

Eg severe neurological symptoms

■ Dead

CIIAPTER 3

Sensitivity of Rabies Virus to TypeI Interferon Is Determined by the

Phosphoprotein Gene: Implications for Viral Pathogenicity

47

Summary

Some studies have shown that P protein of rabies virusinhibits the type 1 interferon

(IFN) signaling pathway. However, the relationship between the P protein function and

viral pathogeniclty remains to be clarified･As described in chapter 2, the author has shown

that the P gene is involved in the difference in pathogeniclty between the avirulentNi-CE

strain and the virulent Ni strain.Hence, the author examined whether the P protein of

Ni-CE strain inhibits the IFN response as does the P protein of Ni strain･ VirulentNi and

CE(Nip) strains that possessed the P gene of Ni strain grew in IFN-treated NA cells and

inhibited the IFN-stimulated response element (ISRE) activity, which is known as an

indicator of the activlty Of the IFN signaling pathway, more efficiently thandid Ni-CE

strain･ Furthermore, the P protein of Ni strain also inhibited the ISRE activlty more

strongly than did the P protein of Ni-CE strain in the absence of other viral proteins･These

results suggest that virulent Ni and CE(Nip) strains acquire higher resistance to IFN than

avirulent Ni-CE strain through inhibition of the IFN signaling pathwayby the P protein･

48

Introductiom

Type I interferon (IFN), such as IFN-α and IFN-ド,has a crucial role in early host

antiviral responses･ Many types of cells rapidly produceIFN in response to viral infections,

and lead the neighboring cells to antiviral state･ The secreted IFN binds toits receptor on

the cell suげace and activates the intracellular IFN signaling pathway consistlng Of many

molecules, such as Janus kinase (JAK) and signal transducer and activator of transcription

(STAT) families. The activated transcription factor translocates to the nucleus, binds to

IFN-stimulated response element (ISRE), and promotes the expression of a number of

proteins with antiviral activity (48)･

To evade the host antiviral responses, many viruses have evolvedto counteract the

IFN-induced actions (48). Recently, it has been reported that P protein of rabies virus

inhibits the IFN signaling pathway by preventing the nuclear accumulation ofSTATl (10,

59). This finding raises the possibility that the function of P protein to inhibit the IFN

slgnaling pathway lS involved in viral pathogenlClty･ However, the relationshipbetween P

protein function and viral pathogeniclty remains to be elucidated･

The author has shown that the substitution rate ofP gene is thehighest among five viral

genes between Ni-CE and Ni strains･ Furthermore, the author has demonstrated thatP gene

is involved in the viral pathogenicity for mice, since chimeric CE(Nip) strains with the P

gene of virulent Ni strain in the background of avirulent Ni-CE strain killed adult mice

after i･c･ inoculation･ Considering the possibility mentioned above, it would beinterestlng

to examine whether the P protein of Ni-CE strain inhibits the IFN responseas does the P

protein of Ni strain･ In this context, the author determined growth e代ciencies ofNi, Ni-CE

and CE(Nip) strains in IFN-treated NA cells. Furthermore, the author determined the ISRE

activities in NA cells infected with each virus and in NA cells transiently expresslng theP

49

protein of Ni and Ni-CE strainsby a reporter assay･ The results suggest that virulent Ni and

CE(Nip) strains acquire higher resistance to IFN than avirulent Ni-CE strain through

inhibition of the IFN signaling pathway by the P protein･

50

Materials and Methods

Cells and viruses

Mouse neuroblastoma NA cells were maintained in Eagle's MEM supplemented with

lO% FCS･ Preliminary experiments showed that NA cells areincompetent to produce type

I IFN but are competent for IFN-induced antiviral response (data not shown)･Therefore,

the author considered NA cells to be suitable for analyzlng theIFN sensitivlty Of each

strain. Ni, Ni-CE and CE(Nip) strains Ⅵ′ere generated by reverse genetics as described in

chapter 2･ Virus stocks of respective strains were preparedin NA cells･

Viral growth in IFN･a･treated NA cells

NA cells grown in a 24-well tissue culture plate (Greiner Bio-One Co･ Ltd･) were

inoculated with Ni, Ni-CE and CE(Nip) strains at a MOI of 0.01. After 1h of virus

adsorptlOn, the NA cells were incubatedin culture medium containlng 0, 20, 100 or

500

units/ml of mouse IFN-α (PBL Biomedical Laboratories, Piscataway, NJ, USA)･The virus

in the culture fluid was harvested at I,3 and 5 dpi and titrated in NA cells

by an

immunofluorescence assay using monoclonal antibody8-1 specific for N protein (39)･The

results of a preliminary experiment confirmed that the concentration ofIFN-α used in this

study did not affect the accuracy of the virus titration (data not shown)･

Plasmids

The PISRE-Luc vector (Stratagene,La Jolla, CA, USA) contains the firefly luciferase

gene downstream of ISRE that is activated through theIFN slgnaling pathway･ The

PRL-TK vector (Promega, Madison, WI, USA), used as an internal controlfor the reporter

assay, contains the Renilla luciferase genedownstream of the herpes simplex virus

5l

thymidine kinase promoter that is activatedin mammalian cells. The PCDNA-Nip and

PCDNA-CEP vectors contain theP gene of Ni and Ni-CE strains, respectively,

in the

expression vector pcDNA3. 1(+) (Invitrogen,Carlsbad, CA, USA)･

Reporter assay

ISRE activities in NA cells infected with each virus andin NA cells transiently

expresslng the P protein of Ni or Ni-CE strains were determinedby a reporter assay･ NA

cells grown in a 24-well tissue culture plate (Greiner Bio-One Co･ Ltd･) were transfected

with 100 ng of PISRE-Luc vector and 20 ng of PRL-TK vector uslngTransIT-Neural

(Mirus). At 24 hr after transfection, the cells were inoculated with Ni, Ni-CE and CE(Nip)

strains at a MOI of 3 and incubated for 6 也.The cells were treated with2000 units/ml of

mouse IFN-α (PBL Laboratories) and then further incubated for 12 h･ Following lysis of

the cells, activities of firefly and Renilla luciferases weredetermined by a Dual-Luciferase

Reporter Assay System (Promega) according to the manufacturer's instructions･ In another

experiment, NA cells were transfected with 250 ng of PCDNA-Nip, PCDNA-CEPor

pcDNA3. 1(+) in addition to the pISREILuc and PRL-TK vectors･ At 24 h after transfection,

the cells were treated with 2,000 units/ml of IFN-α andincubated for 6 h. After preparation

of the cell lysate, activities of firefly and Renilla luciferases weredetermined as described

above･ ISRE activlty Was Calculated by normalizlng the activlty Offirefly luciferase to the

activity Of Renilla luciferase･

52

Results

Growth of Ni-CE and Ni strains in IFN･treatedNA cells

First, to examine whether the IFN sensitivlty Of virulentNi strain is different from that

of avirulent Ni-CE strain, the author compared the viral growthin NA cells treated with

100 units/ml of IFN-cx. In the absence ofIFN-α, both Ni and Ni-CE strains efficiently

grew to a titer of about 107 focus-forming units (FFU)/ml (Fig･ 3-1)･ When NA cellswere

treated with IFN-α, the virus titer of Ni strain gradually lnCreaSed and reached about105

FFU/ml at 5 dpi･ In contrast, the virus titer of Ni-CE strain hardlyincreased (about 102

FFU/ml at 5 dpi).

Next, to confirm the reproducibility of the phenomenon described above, the author

treated the infected cells with 0, 20, 100 and 500 units/ml ofIFN-α, respectively, and

determined the virus titer in the culture fluid at 5 dpi･ While the virus titer ofNi strain

gradually decreased according to the dose of IFN-α, it Ⅵ′as more than105 FFU/ml even in

cells treated with 500 units/ml of IFN-α (Fig. 3-2). In contrast, the virus titer of Ni-CE

strain drastically decreased and was below 102 FFU/ml in cells treated with500 units/ml of

IFN-α. These results demonstrate that the avirulent Ni-CE strainis more sensitive to

IFN-induced antiviral responses than is the parental virulent Ni strain･

Growth of CE(Nip) strain in IFN-treated NA cells

To examine whether the P gene is involvedin the difference between IFN sensitivities

of Ni and Ni-CE strains, we evaluated the IFN sensitivity of the chimeric CE(Nip) strain･

In the absence of IFN-α, the virus titer of CE(Nip) strain in the culture fluid reached about

107 FFU/ml, which was comparable to virus titers of Ni and Ni-CE strains (Fig･ 311)･In the

presence of 100 units/ml oflFN-α, the virus titer of CE(Nip) strain reached about 105

53

FFU/ml as did the Ni strain, which was a markedly higher titer than that of Ni-CE strain.

When the cells were treated with various dose of IFN-α, the virus titer of CE(Nip) strain

gradually decreased dose-dependently'but it was more than 104 FFU/ml even in cells

treated with 500 units/ml of IFNICL (Fig. 3-2). These results indicate that the IFN

sensitivlty is determined by the P gene of the virus･

ISRE activities in NA cells infectedwith Ni, Ni-CE and CE(Nip) strains

The different IFN sensitivities among Ni, Ni-CE and CE(Nip) strains may be caused by

the ability to inhibit the IFN signaling pathway･ To test this possibility, the author

examined the ISRE activlty ln Virus-infected NA cells, whichis activated via the IFN

slgnaling pathway･ In mock-infected cells, ISRE activlty Was markedly lnCreaSedin

response to IFN-α treatment (Fig. 3-3).Similarly, ISRE activity in NA cells infected with

Ni-CE strain was clearly lnCreaSed･ In contrast, ISRE activitiesin NA cells infected with

Ni and CE(Nip) strains increased to only half the level of that in mock-infected cells in

response to IFN-α treatment. These results indicate that virulent Ni and CE(Nip) strains

inhibit the IFN-induced activation of the ISRE promoter.

ISRE activities in NA cells expresslng the P protein of Ni and Ni-CE strains●

To examine whether the inhibition of ISRE activlty ln infected cells is caused by the P

protein of the virus, the author determined the ISRE activities in NA cells transiently

expresslng the P protein of Ni or Ni-CE strains･ ISRE activlty in NA cells transfected with

an empty vector markedly increased in response to IFN-α treatment (Fig･ 3-4)･In NA cells

expresslng the P protein of Ni-CE strain, the ISRE activlty alsoincreased in response to

IFN-α treatment, although the level was slightly lower than that in empty

54

vector-transfected cells. On the otherhand, in NA cells expresslng the P protein of Ni

strain, the ISRE activlty ln the presence of IFN-α was less than half that in empty

vectoトtranSfected cells. These results indicate that the P protein of virulent Ni strain more

effectively inhibits the IFN-induced activation of the ISRE promoter than does the P

protein of avirulent Ni-CE strain in the absence of other viral proteins･

55

Discussion

The author examined whether the P proteins of virulent and avirulent strains inhibit the

IFN response. Virulent Ni and CE(Nip) strains that possessed the P gene of Ni strain grew

in IFN-treated NA cells and inhibited the ISRE activlty, Which is known as an indicator of

the activlty Of the IFN signaling pathway, more efficiently than did avirulent Ni-CE strain･

Furthermore, the P protein of Ni strain also inhibited the ISRE activlty more Strongly than

did the P protein of Ni-CE strain in the absence of other viral proteins. These results

suggest that virulent Ni and CE(Nip) strains acquire higher resistance to IFN than avirulent

Ni-CE strain through inhibition of the IFN signaling pathway by the P protein.

It has been reported that the P proteins of some other laboratory strains of rabies virus

interact with STATl and inhibit the IFN signaling pathway by preventlng STATl nuclear

accumulation (10, 59). Therefore, the difference in the P protein function between Ni and

NトCE strains to inhibit the ISRE activlty may be caused in this process. The importin α5

mediating the nuclear transport of STATl binds to nuclear localization~ signal (NLS) in the

DNA binding domain of STATl (36). Also, the P protein binds to around the DNA

binding domain of STATl (59),suggesting that the P protein masks the NLS by interacting

with STATl and interfering with the interaction betⅥ′een STATl and importin α5. There

were five amino acid substitutions in the P protein between Ni and Ni-CE strains, of which

four were all changed to proline, which generally affects protein structure. Therefore, these

amino acid substitutions in the P protein of Ni-CE strain may cause structural change, alter

the affinity of P protein for STATl and impalr the function toinhibit the interaction

between STATl and importin (15.

The P protein of rabies virus also has an NLS (residues 211 to 214 and 260) and is

transported to the nucleus by an undetermined cellular transport factor (43)･This fact raises

56

the possibility that the P protein interacts with importin α5 via the NLS and competitively

inhibits the nuclear transport of STATl･ Interestlngly, amino acid substitution at position

226 in the P protein between Ni-CE and Ni strains is structurally close to the NLS and the

putative phosphorylation site (atposition 210) (17), as shown in the crystal structure of P

protein (35). Some studies have shown that the phosphorylation state around the NLS

modulates the efficiency of nuclear transport of the protein (21, 65). Therefore, the amino

acid substitution at position 226 in the P protein of Ni-CE strain may alter the

phosphorylation efficiency and the affinity of P protein for importin α5 and reduce the

inhibitory effect on the nuclear transport of STATl.

Meanwhile, it is possible that the P protein inhibits the IFN signaling pathway ln the

nucleus. IFN-stimulated gene factor 3 (ISGF3), a complex of STATl, STAT2 and an IFN

regulatory factor family p48, binds to ISRE and promotes the expression of IFN-inducible

genes. As mentioned above, the P protein binds to around the DNA binding domain of

STATl, which is important for the binding of ISGF3 to ISRE (4)･Therefore, the P protein

may lnterrupt the interaction between ISGF3 and ISRE and prevent the expression of

IFN-inducible genes.

STATl has a nuclear export signal (NES) for returning to the cytoplasm, which is

thought to be important for maintaining the functionality of the IFN signaling pathway (37)･

The nuclear export of STATl is mediated by chromosome region maintenance 1 (CRMl),

a member of the family of cellular transport factors (37). Notably, the P protein also has a

CRMl-dependent NES (residues 49 to 58) (43), suggesting that the P protein interferes

with the recycling of STATl･ Interestlngly, amino acid substitutions at positions 56 and 58

in the P protein between Ni-CE and Ni strains were located in important residues of the

NES (32). Therefore, it is possible that these amino acid substitutions disrupt the

57

interaction between the P protein and CRMl andimpalr the ability to inhibit the recycling

of STATl･ Taken together, the author speculates that theP protein inhibits various

processes of the IFN signaling pathwayln the cytoplasm and nucleoplasm･

Here, the author showed that the virulent CE(Nip) strain is less sensitive to type I IFN

than is the avirulent Ni-CE strain･ Interestlngly, it has been reported that type I IFNis

produced in the mouse brain in response to rabies virus infection (28, 60)･ Therefore, it is

thought that the different IFN sensitivities of Ni-CE and CE(Nip) strains influence their

propagation efficiencies in vivo and result in their different pathogenicities･ Infact, the

author found that the titer of CE(Nip) strain in the adult mouse brain reaches 105 FFU/g,

whereas that of Ni-CE strain is less than 102 FFU/g at 3 days after i･c･ inoculation of 100

FFU of each virus (data not shown).

As described in chapter 2, CE(NiN) and CE(NiM) strains were also virulent, although

these strains possessed the P gene of NトCE strain･ Hence, the author speculates that the N

and M genes are involved in viral pathogenlCltyln another fashion, respectively･ In order

to fully understand the attenuation mechanism of Ni-CE strain,fu血er study will be

needed to elucidate the roles in pathogeniclty Of N and M genes･

1n conclusion, the author has shown for the first time that an avirulent strain of rabies

virus is more sensitive to type I IFN than is a virulent strain and that IFN sensitivlty Of the

virus is associated with P protein function to inhibit the IFN signaling pathway･ For

elucidation of the detailed mechanism, it is necessary to determine which processes

become targets for the P protein and which amino acids in the P protein areimportant for

the function. In addition, further work is required to clarify the relationshipbetween

pathogeniclty and IFN sensitivlty Of rabies virus in vivo･

58

Legends to figures

Fig･ 3･1･ Viral growth in NA cells treated withIFN-α･ NA cells were inoculated with Ni,

Ni-CE and CE(Nip) strains at a MOI of 0.01 and incubated in culture medium with (open)

or without (filled)IFN-α (100 U/ml). The virus in the culture fluid was harvested at 1,3,

and 5 days post-inoculation and titrated in NA cells･

Fig･ 3-2･ Viral growth in NA cells treated with various doses of IFN-α･ NA cells were

inoculated with Ni, Ni-CE, and CE(Nip) strains at a MOI of 0.01 and incubatedin culture

medium with 0, 20, 100, and500 U/ml of IFN-α･ The virus

in the culture fluid was

harvested at 5 days post-inoculation and titratedin NA cells･

Fig. 3･3. ISRE activities in NA cells infected withNi, Ni-CE and CE(Nip) strains･ NA

cells were transfected with reporter plasmids, pISREILuc and pRLITK･At 24 h after

transfection, the cells were inoculated with each strain at aMOI of 3 and further incubated

for 6 h. Then the cells were treated with2,000 U/ml of IFN-α for 12 h and subjectedto

luciferase assay･ ISRE activlty lS expressed as the ratio offirefly luciferase activlty tO

Renilla luciferase activlty･ Error bars indicate standard deviations of the ratio･Asterisks

indicate significant difference between the values (P < 0･01)I

Fig･ 3･4･ ISRE activities in NA cells transiently expresslng theP protein of Ni and Ni-CE

strains･ Reporter plasmids were transfected to NA cells together with pcDNA3･1,

PCDNA-Nip or PCDNA-CEP･ At 24 h after transfection, cells were treated with2,000 U/ml

of IFN-α for 6 h and s叫ectedto luciferase assay･ ISRE activlty lS expressed as the ratio

of firefly luciferase activity tO Renillaluciferase activlty･ Error

bars indicate standard

59

deviations of the ratio. Asteriskindicates significant difference between the values (P <

0.01).

60

CE(Nip)

1 3 5

Ni-CE

1 3 5

Days post-inoculation

Fig. 311

61

Ni

/8

6

4

2

育=)LL[エー

bJ)○

∴

＼■_′

iZ

q)

･≡(′〕

コ

.t=>

8

7

育=)【.L[エ■

bJ)○

--

＼__′

喜≡

qJ

･=∽

コ

.t:>

6

5

4

3

20 100 500

IFN-α (U/ml)

Fig. 3-2

62

■Ni

田Ni-CE

□ cE(Nip)

Ni Ni-CE CE(Nip)

Fig. 3-3

63

20

>ヽ

.亡

.己15巴=コO

cd

岩10(′つ■･･･････■

5

pcDNA3･1 PCDNA-Nip PCDNA-CEP

Fig. 3･4

64

IFN-α

(2000 U/ml)

[コー

■ +

CONCLUSIONS

l･ N, P and M genes are related to the difference in pathogeniclty between Ni-CE and Ni

strains.

2.. The attenuation mechanism of Ni-CE strain is different from that of avirulent RCIHL

strain, which is also derived丘･om Ni strain.

3. Ni-CE strain is more sensitive to type I IFN than is Ni strain･

4･ The P gene of rabies virus determines IFN sensitivlty Of the virus･

5･ The P protein of Ni strain inhibits the IFN signaling pathway more efficiently than does

the P protein of Ni-CE strain in the absence of other viral proteins.

These findings provide useful information for the development of improved live vaccines

and therapeutic measures.

65

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to Professor Dr･ Makoto Suglyama, United

Graduate School of Veterinary Sciences, Gifu Universlty, for invaluable discussion and

comprehensive support for completion of this thesis･ I am deeply grateful to Honorary

Professor Dr. Nobuyuki Minamoto, Gifu Uniyerslty, for kind direction, encouragement and

constructive advice. I express my appreciation to Associate Professor Dr･ Naoto lto,

Laboratory of Zoonotic Diseases, Faculty of Applied BiologlCal Sciences, Gifu Universlty,

for continulng Support and fruitful discussion. 1 am grateful to Professor Dr･ Hiroshi

Suzuki, Obihiro Universlty Of Agriculture and Veterinary Medicine, Professor Dr.

Kunihiro Sbinagawa, Iwate Universlty, Professor Dr. Eiichi Honda, Tokyo Universlty Of

Agriculture and Technology, and Professor Dr. Hideto Fukushi, Gifu Universlty, for

valuable comments and criticisms. I am thankful to Dr. S. Makino, Universlty Of Texas

Medical Branch, U.S.A., for helpful technical support. Finally, I sincerely thank members

of Laboratory of Zoonotic Diseases, Faculty of Applied BiologlCal Sciences, Gifu

Universlty, for their various supports.

66

REFERENCES

1) Albertini, A.A., Wernimont, A.K･, Muziol, T･, Ravelli, R･B･, Clapier, C･R･, Schoehn,

G., Weissenhorn, W. and Ruigrok, R･W･ (2006)･ Crystal structure of the rabies virus

nucleoprotein-RNA complex･ Science 3 13(5785), 360-363･

2) Black, J.G. and Lawson, K.F. (1980). The safety and efficacy of immunizing foxes

(Vulpes vulpes) usingbait containing attenuated rabies virus vaccine･ Can･ J･ Comp･

Med. 44, 169-176.

3) Blondel, D., Regad, T., Poisson, N., Pavie, B･, Harper, F･, Pandolfi, P･P･, De The, H･

and Chelbi-Ali又, M.K. (2002). Rabies virus P and small P products interact directly with

PML and reorganize PML nuclear bodies･Oncogene 21(52), 7957-7970･

4) Bluyssen, H.A. and Levy, D.E. (1997)･ STAT2 is a transcriptional activator that

requlreS Sequence-Specific contacts provided by STATl and p48 for stable interaction

with DNA. ∫.Biol. Cbem. 272(7), 4600-4605.

5) Bonilla, W.Ⅴ., Pinschewer, D.D., Klenerman, P., Rousson, V., Gaboli, M･, Pandolfi,

P.P., Zinkernagel, R.M., Salvato, M.S. and Hengartner, H･ (2002)･ Effects of

promyelocytic leukemia protein on virus-hostbalance･ J･ Virol･ 76(8), 3810-3818･

6) Bourhy, H., Sureau, P. and Tordo, N. (1990)･ From rabies to rabies-related viruses･

Vet. Microbiol. 23(1-4), 1 15-128.

7) Briggs, D.∫., Dreesen, D.W. and Wunner, W.H. (2002)･ Vaccines･ In: Jackson, A･C･

and Wunner, W.H.