Elucidation of Attenuation Mechanism of the Ni-CE Strain of Rabies Virus Established from Nishigahara Strain by Passages in Chicken Embryo Fibroblast Cells

Title

Elucidation of Attenuation Mechanism of the Ni-CE Strain of

Rabies Virus Established from Nishigahara Strain by Passages in

Chicken Embryo Fibroblast Cells( 本文(Fulltext) )

Author(s)

清水, 健太

Report No.(Doctoral

Degree)

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)

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)

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

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),}

patients develop 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 used for

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

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

continuously been a _{serious problem･} The facts described _above are _{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 _vaccines can _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. Itwas _{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

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 can be

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)

showed by generating a _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 strain is 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 isnot _{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

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)

_related to _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 pathway} by the P

protein･ These findings provide useful information for the development of improved live

CIIAPTER

1

Comparison

_{of the Complete}

Genome

Sequence

_{of Avirulent}

Ni-CE

Strain

_{of Rabies}

Virus

_Ⅵ7ith

That

_or

the

Parental

Virulent

Summary

Rabies _{virus Ni-CE strain} causes _nonlethal infection in adult mice after i.c. inoculation,

whereas the parental Ni strain kills mice･ To clarify the genetic differences 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 of Ni-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 were identical 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 was O･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 of NトCE strain is simpler than that of RC-HL strain･ A previous study demonstrated that RC-HL and Ni _strains have the lowest _sequence homology ln _{the G gene among} five _{viral genes･} In

contrast, the author showed that the P gene has the highest _substitution rate at both

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 the difference in pathogeniclty between Ni and RC-HL strains were all conserved between Ni-CE and Ni strains･ These findings suggest that the attenuation mechanism of Ni-CE strain is different from that of RC-HL strain.

Introduction

The _{genome of} rabies virus is a _{nonsegmented negative-sense} RNA _composed of

approximately 12,000 nucleotides･ It encodes five structural proteins: N, P, M, G and L proteins･ The N, P and L proteins form 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 derived from 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 the induction of virus neutralizing antibody

(64)･

The fixed _{rabies viruses} are _classified into virulent and avirulent strains by pathogenlClty for mice: virulent strains kill adult mice after i･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 the G 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 _responsible for the viral

pathogenicity. These findings support the results of previous studies

(13,

51,

58)

showing that the G protein isa _major determinant _{of the pathogeniclty} Of rabies virus･ On the other

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, itwas

shown that chimeric viruses with the G gene and atleast one _{other gene} from 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 _and Ni _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.}

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 _{strain did}

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

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).

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 were no

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 and Ni strains

are _shown in Tableト3. The _{nucleotide substitution} rate _{of the P gene}

_(1.12%)

was higher

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 _{all lower}

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 proteins} between Ni-CE

and Ni strains are _shown in Fig･ 1-3･ A total of 15 amino acid substitutions were found in

various reglOnS･ In the N protein, three amino acid substitutions were found 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 located in the amino-terminal reglOn and one in the central reglOn･ The amino acids at positions 242, 255, and 268 in the G protein, which were _shown to be related to the difference in pathogeniclty

Discussiom

To _{clarify the genetic} differences between _{the avirulent} NトCE _{strain and the parental}

virulent Ni strain, the author determined the complete genome sequence of Ni-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 should be noted that amino acids at _positions 242, 255 _and 268 in the G _protein, which were identified as

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 position 333 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 of Ni-CE strain is different from _{that of RC-HL strain} as _well as _{many other strains･}

The Ni-CE _strain was _genetically more _conserved than RC-HL strain when compared

to _{the parental Ni strain, especially in the G gene, which} is important for entry of the virus

into host _{cells･ This} difference _{is probably} due to _passage histories _{of these strains: Ni-CE}

strain Ⅵ′as _established from Ni strain by 100 passages only in 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}

(at

positions 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

_adjacent

to 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

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

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}

_{(1 1)･}

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 background between Ni-CE _and Ni _{strains, and the author} has _{also shown the possibility} that Ni-CE strain is attenuated by a _{novel mechanism.}

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

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･

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 1 474- 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.}

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.

g + h-1 + u 二二 e + f+ Fig. 1･1

3ー 11926 ^L 5ー 1r l35389460915756384 ^L (297) P (202) M .JL ..一L 1r (450) 1r (524) 山llllll■■■■一山(2127) N G L 70 91 88 211 520 131 Fig. 1･2

☆

☆

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 2127 aa

CHAPTER

2

Involvement

_{or Nucleoprotein,}

Phosphoprotein

_and

Matrix

Protein

Genes

_{of Rabies}

Virus

in Virulence

for Adult

Mice

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.}

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 protein are _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 333 in 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 of four}

amino acid substitutions, all of which were _changed to _{proline residue, which} is thought to

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

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.}

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}

addition to the Pstl site used as a _{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

treated _with restriction endonuclease 〟/〟I and electrophoresed on _{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. Five 2-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

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 then increased･ The body _weight curve _of

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 itwas 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 titerat 1 dpi was comparable to

others.

Pathogenicity _{of chimeric viruses in mice}

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 other hand,

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 mice} inoculated _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 lose body 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 mice} inoculated _with

CE(NiN),

CE(Nip)

_and

CE(NiM)

strains developed severe _{neurologlCal symptoms and} died _{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.

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}

Discussion

To identify _viral

_gene(s)

_related to 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 the difference 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 transcription 1

(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 the host} defense _mechanism

(5, 14)･

Although the _{slgnificance of these} functions _{of the} P _{proteins of rabies virus in vivo is} unclear, the author speculates that these functions a什ect the differences 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 the functional motif required for the nucleocytoplasmic transport of P protein

(43),

as described in

chapter 1: amino acid substitutions at positions 56 and 58 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

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 patbogeniclty} lS _{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 inhibit host

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 the impalrment 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

_(residues

89 to

_loワ)(56),

_which is thought to _{be associated with}

the host _membrane･ Therefore, _changes in these amino acids may affect the interaction