Advances in Virus Research

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 CONTENTS

Poliovirus Assembly and Encapsidation of Genomic RNA

DAVIDANSARDI.DONNAC. PORTER. MARIEJ . ANDERSON.
AND CASEYD . MORROW
I. Overview ........................................................ 2
I1. Genomic Organization ............................................ 3
I11. Poliovirus Life Cycle ............................................. 6
IV . Poliovirus Virion ................................................. 14
V. Morphogenesis of Poliovirus ...................................... 19
VI . RNA Encapsidation Process ....................................... 30
VII . New Methods to Study Poliovirus Assembly ........................ 34
VIII . Complementation System to Study Poliovirus Encapsidation ........ 39
IX . Perspectives on Poliovirus Assembly ............................... 53
References ....................................................... 56

Genome Rearrangements of Rotaviruses

ULRICH DESSELBERGER
I. Discovery of Genome Rearrangements ............................. 71
I1. Extent of Genome Rearrangements in Rotaviruses ................. 75
I11. Sequence Data of Rearranged Genes ............................... 75
IV . Genome Rearrangements Generated in Vitro ....................... 79
V. Mechanisms of Genome Rearrangements .......................... 82
VI . Biophysical Data ................................................. 86
VII . Function of Rearranged Genes and Their Products ................. 86
VIII . Genome Rearrangements and Evolution of Rotaviruses ............. 91
IX . Genome Rearrangements in Other Genera of Reouiridae ............ 92
X. Outlook ......................................................... 92
References ....................................................... 93

Human ImmunodeficiencyVirus Type 1 Reverse Transcriptase

and Early Events in Reverse Transcription
ERICJ . ARTSAND MARKA . WAINBERG
I. Introduction ..................................................... 99
I1. Overview of Human Immunodeficiency Virus Type 1 Replication .... 101
V
vi CONTENTS

I11. Human Immunodeficiency Virus Type 1 ........................... 107

IV . Human Immunodeficiency Virus Type 1 Reverse Transcription ...... 119
References ....................................................... 146

Hepadnaviruses: Current Models of RNA Encapsidation

and Reverse Transription

A . FALLOWS
DOROTHY P. GOFF
AND STEPHEN
I. Introduction ..................................................... 167
I1. Transcription and Translation .................................... 172
I11. RNA Encapsidation .............................................. 176
IV . The Hepadnaviral Polymerase .................................... 180
V. Reverse Transcription ............................................ 184
VI . Concluding Remarks ............................................. 192
References ....................................................... 193

Cell Types Involved in Replication and Distribution

of Human Cytomegalovirus

BODOPLACHTER. SINZGER.
CHRISTIAN AND GERHARD
JAHN
I. Introduction ..................................................... 197
I1. Determinants of Human Cytomegalovirus ......................... 198
I11. Organ Tropism of Human Cytomegalovirus ........................ 216
IV . Cells Types Involved in Acute Human Cytomegalovirus Disease ..... 219
V. Viral Spread and Pathogenesis .................................... 232
VI . Latent Cytomegalovirus Infection ................................. 236
VII . Summary ........................................................ 241
References ....................................................... 241

Varicella-Zoster Virus: Aspects of Pathogenesis and the Host Response

to Natural Infection and Varicella Vaccine

ANN M . ARVIN.JENNIFER F . MOFFAT. AND REBECCA REDMAN

I. Introduction ..................................................... 265
I1. The Virus ....................................................... 266
I11. Cell-Associated Viremia in the Pathogenesis of Varicella-Zoster
Virus Infection ................................................... 267
IV. The Cell-Mediated Immune Response to Varicella-Zoster Virus ...... 280
V. Summary ........................................................ 306
References ....................................................... 307

Anatomy of Viral Persistence: Mechanisms of Persistence

and Associated Disease

JUANCARLOS
DE LA TORREAND MICHAELB . A . OLDSTONE
I . Introduction ..................................................... 313
I1. Requirements for Establishment of Viral Persistence ............... 315
 CONTENTS vii

111. Virus-Induced Alterations of Host Cellular Differentiated Functions

in Absence of Cytolysis ........................................... 323
IV . Conclusions ...................................................... 338
References ....................................................... 340

The lridoviruses

TREVOR
WILLIAMS
I. Introduction ............. ........................... 347
I1. Classification .................................................... 350
I11. Structure ................... ...................... 366
IV . Replication ...................................................... 372
V. Molecular Biology ................................................ 386
VI . Ecology ...................... ..................... 391
VII . Future Directions for Iridoviruses ................................. 399
References .............. ..................... 401

Molecular Biology of Luteoviruses

M . A . MAYOAND V . ZIEGLER-GRAFF
I . Introduction .................. .......................... 416
I1. Genome Structure ................................................ 417
111. Functions of Gene Products ....................................... 424
IV . Mechanisms of Gene Expr ................... 435
V. Particle Structure ................................................ 444
VI . Location of Luteovirus Replication ....................... 449
VII . Phytopathology .................................................. 450
VIII . Taxonomy .............. 453
IX . Concluding Remarks . . . . 457
References .... ................................... 457

INDEX ........................................................... 463

This Page Intentionally Left Blank
 t

POLIOVIRUS ASSEMBLY AND ENCAPSIDATION

OF GENOMIC RNA

David C. Ansardi, Donna C. Porter, Marie J. Anderson,

and Casey D. Morrow

Department of Microbiology
University of Alabama at Birmingham
Birmingham, Alabama 35294

I. Overview
11. Genomic Organization
111. Poliovirus Life Cycle
A. Virus Entry and Uncoating
B. Translation of Viral RNA
C. Release of Individual Proteins by Viral Proteases
D. Replication of Viral RNA
IV. Poliovirus Virion
A. Properties of Virion
B. Virus Structure
C. Myristylation of Poliovirus Capsid Proteins
V. Morphogenesis of Poliovirus
A. 5s Protomer
B. 14s Pentamer
C. Empty Capsid
D. Provirion
VI . RNA Encapsidation Process
A. RNA Requirements for Encapsidation
B. Poliovirus Defective Interfering Particles
C. RNA Encapsidation Signals
D. Subcellular Location of Encapsidation
VII. New Methods to Study Poliovirus Assembly Process
A. Studies of Poliovirus Assembly Process Using Recombinant
Vaccinia Viruses
B. Expression of Poliovirus P1 and 3CD Using Recombinant Vaccinia
Virus Vectors
C. Functional Significance of Poliovirus Capsid Myristylation
VIII. Complementation System to Study Poliovirus Encapsidation
A. Proteolytic Cleavage of Capsid Precursor
B. Capsid Mutations Affecting RNA Encapsidation
C. Studies on Maturation Cleavage Using Complementation System
IX. Perspectives on Poliovirus Assembly
References

1
Copyright 0 1996 by Academic Press,Inc.
All rights of reproduction in any form resewed.
2 DAVID C. ANSARDI et al.

I. OVERVIEW

The biology of poliovirus has been a subject of intense study since

the 1950’s. Poliovirus is the causative agent of the paralytic disease
poliomyelitis, once a major health problem in the United States that
has largely been eradicated since the development of two highly effec-
tive vaccines (Sabin and Boulger, 1973; Salk, 1960). Despite control of
the disease in industrialized nations, poliomyelitis continues to be a
health concern in the undeveloped world.
Poliovirus is a member of a family of viruses, the Picornauiridae,
that includes members responsible for several diseases of humans,
including the human rhinoviruses (common cold), hepatitis type A,
and the coxsackieviruses (cardiac infections) (Rueckert, 1990). Other
members of the Picornauiridae are responsible for important diseases
of livestock, including foot-and-mouth disease virus, bovine entero-
virus, and the causative agent of swine vesicular disease. Another
group of picornaviruses, the cardioviruses, primarily infect mice and
includes members such as mengo virus and encephalomyocarditis vi-
rus (EMCV). Poliovirus, like all of the members of the Picornauiridae,
is a spherical, single-stranded RNA virus. The viral genome is a n
approximately 7500-nucleotide-long RNA molecule of positive polarity
(messenger-sense) and is encapsidated within a virion particle that is
approximately 30 nm in diameter (Kitamura et al., 1981; Koch and
Koch, 1985). The poliovirus genome has been cloned and sequenced
(Kitamura et al., 1981; Racaniello and Baltimore, 1981a1, greatly facil-
itating the analysis of specific proteins and cis-acting regions of the
RNA genome in the life cycle of the virus.
Three different antigenically distinct serological types of virulent
poliovirus have been identified, designated types 1 , 2 , and 3 (Koch and
Koch, 1985). The polioviruses are members of the enterovirus genus of
the Picornauiridae,and as such primarily inhabit the alimentary canal
of the host. Most infections with poliovirus do not result in paralytic
disease. However, in some instances, poliovirus spreads from the intes-
tine to the central nervous system; lytic replication of the virus in
motor neurons results in paralysis, which can often be fatal (Koch and
Koch, 1985). Control of poliovirus infection in modern nations is large-
ly based on the success of a highly effective oral vaccine consisting of
live, attenuated strains of poliovirus (Sabin and Boulger, 1973). The
attenuated strains given to the vaccine recipient replicate in the intes-
tine, where they stimulate immunity against poliovirus infection, but
are incapable of causing paralytic disease. The highly effective nature
of the poliovirus vaccines has led to intensive research into the applica-
tion of poliovirus as a vector for delivering foreign antigens to the
 POLIOVIRUS ASSEMBLY AND RNA ENCAPSIDATION 3

immune system, opening the possibility that this human pathogen

might be harnessed for helpful purposes (Almond and Burke, 1990;
Ansardi et al., 1994b; Porter et al., 1993a, 1995).
Several developments have made poliovirus an excellent model for
studying the molecular processes of viral replication. Poliovirus can be
grown in many tissue culture cell lines of human and primate origin
(Koch and Koch, 1985). The viral genome has been cloned and se-
quenced, revealing the nucleotide sequence of the RNA genome and
the predicted amino acid sequences for the viral proteins (Kitamura et
al., 1981; Racaniello and Baltimore, 1981a). cDNA copies of the po-
liovirus RNA genome are infectious and result in a productive virus
infection on transfection into suitable host cells (Racaniello and Bal-
timore, 1981b; Semler et al., 1984). The infectivity of poliovirus cDNA
has allowed the use of techniques such as site-specific mutagenesis to
alter the coding sequence of the virus (Zoller and Smith, 1983). A
further advance was made with the finding that positive-sense RNA
genomes transcribed in uitro from poliovirus cDNA, under the control
of the promoter for bacteriophage T7 RNA polymerase, were highly
infectious on transfection into host cells (Van der Werf et al., 1986).
In 1985, the three-dimensional structure of poliovirus was solved,
providing detailed information about the structure of poliovirus capsid
proteins and insight into possible mechanisms of poliovirus morpho-
genesis (Hogle et al., 1985). The cell surface protein receptor used
by poliovirus to gain entry into the host cell has been cloned and
sequenced (Mendelsohn et al., 1989). Transgenic mice which express
the poliovirus receptor have also been generated, providing an animal
model in which the molecular mechanisms of poliovirus pathogenesis
can be studied (Ren et al., 1990).

11. GENOMICORGANIZATION

The organization of the poliovirus RNA genome and the cascade of

the formation of individual viral proteins are presented in Fig. 1. The
positive-sense RNA genome of poliovirus is 7441 nucleotides in length
(Kitamura et al., 1981; Racaniello and Baltimore, 1981a). The 5' end of
the RNA genome is not linked to a 7-methylguanosine cap, but instead
is covalently linked to a virus-encoded basic peptide of 22 amino acids,
known as VPg (genome-linked protein), through a phosphodiester link-
age between the 0 4 hydroxyl group oxygen of a tyrosine residue in
VPg and the phosphate of the 5' terminal uridine residue of the RNA
genome (Ambros and Baltimore, 1978; Lee et al., 1977; Morrow et al.,
1984; Nomoto et al., 1976; Rothberg et al., 1980; Wimmer, 1982).The 3'
4 DAVID C. ANSARDI et al.

5, VPg-IRESy W APSID-NON-C
33;86
APSI-
7370~ 7 4 4 1
AA(AEnAA 3'
OPEN READING FRAME n=dO

POLYPROTEIN
A A

-PLF
1[yp3lpiq
?+N.A.+PO
A P qm p q 2BC 3AB

4 m VP4
MlzC1ElO38 uncleaved
3C + 3D

(VPg)
A cleavage catalyzed by 2A A cleavage catalyzed by 3CD
A cleavage cntdyzd by 3C A ~ ~ ~ ~ ; ~ ~ ~ ~ ~ l , w n

FIG.1. Poliovirus genomic organization and cascade of polyprotein processing. The

poliovirus genome is a single-stranded messenger (plus sense) RNA molecule that is
approximately 7500 bases in length. The 5' end of the RNA molecule is covalently linked
to a small peptide, VPg, and the 3' end contains a genetically encoded polyadenylate tail
that is approximately 60 nucleotides long. The first 742 nucleotides at the 5' end of the
genome comprise the 5'-N"R, which contains the internal ribosome entry sequence
(IRES). The poliovirus genome contains a single open reading frame encoding a 2209-
amino acid polyprotein precursor. Virus-encoded proteases 2A and 3C catalyze cis-acting
cleavages of the polyprotein to initiate the cascade of formation of the individual viral
proteins. Further processing of the viral proteins is primarily mediated by 3C, although
the 3CD polyprotein catalyzes cleavage of the P1 capsid precursor to VPO, VP3, and VP1.
Both 3C and 3CD catalyze cleavages a t glutamine-glycine dipeptides, whereas 2A cata-
lyzes cleavages between tyrosine-glycine amino acid pairs. The final cleavage event
occurs at an asparagine-serine amino acid pair on the interior of the virion, resulting in
conversion of VPO to VP2 and VP4. The source of this cleavage is unknown, but it is
speculated to occur intramolecularly.

end of the genome is polyadenylated, with a tail length of approx-

imately 60 adenine residues. The poly(A) tail is genetically encoded by
the virus rather than added by host cell polyadenylation enzymes
(Kitamura et al., 1981; Racaniello and Baltimore, 1981a; Spector and
Baltimore, 1975; Yogo and Wimmer, 1975). The majority of the viral
RNA genome (6627 nucleotides) constitutes a long open reading frame
that encodes a single translation product of 2209 amino acids. An
unusually long nontranslated region of 742 nucleotides (5'-NTR) pre-
 POLIOVIRUS ASSEMBLY AND RNA ENCAPSIDATION 5

cedes the open reading frame upstream of the initiation codon used for
translation of the genomic polyprotein. The 5'-NTR contains eight
AUG triplets prior to the one which actually serves as the initiation
codon for translation of viral proteins (Kitamura et al., 1981; Pelletier
et al., 1988; Racaniello and Baltimore, 1981a). Translation of po-
liovirus as well as other picornavirus RNA genomes occurs by a cap-
independent method, in which ribosome binding occurs at an internal
sequence known as the internal ribosome entry sequence (IRES) (Jang
et al., 1988; Pelletier et al., 1988; Pelletier and Sonenberg, 1988; Sonen-
berg, 1990; Trono et al., 1988).
The coding portion of the poliovirus genome is subdivided into three
distinct regions, designated P1, P2, and P3 (Kitamura et al., 1981;
Rueckert and Wimmer, 1984). The P1 region encodes the viral capsid
proteins VP1, VP2, VP3, and VP4. The P2 region encodes nonstruc-
tural viral proteins including a protease, 2A, and 2B and 2C, which
are believed to play roles in replication of the RNA genome. The P3 re-
gion encodes nonstructural proteins required for virus replication,
including 3Dpo1, the RNA-dependent RNA polymerase, a protease,
3Cpr0, and the VPg protein (also known as 3B). Many of the viral pro-
teins have important functions in polyprotein forms; for example, the
membrane-bound 3AB protein is a component of the replication com-
plex (Giachetti and Semler, 1991; Semler et al., 1982), and the 3CD
polyprotein catalyzes proteolytic cleavages of the capsid precursor
(Jore et al., 1988; Ypma-Wong et al., 1988a).
All of the proteolytic cleavages required to liberate individual po-
liovirus proteins required for replication and encapsidation of the ge-
nomic RNA are catalyzed by virus-encoded proteases which cleave the
primary translation product both in cis and in trans (Dewalt and Sem-
ler, 1989; Hanecak et al., 1982; Harris et al., 1990; Lawson and Semler,
1990; Palmenberg, 1990; Toyoda et al., 1986). The primary cleavage of
the genomic polyprotein is an intramolecular event in which the 2A
protease processes the peptide bond between a tyrosine-glycine di-
peptide, releasing the 97-kDa polyprotein encoded by the P1 region
(Toyoda et al., 1986). The P1 protein is a precursor from which the
individual capsid proteins of the virus are derived. The virus-encoded
protease 3Cpr0, acting in a polyprotein form, 3CD, is responsible for
cleavage of the P1 precursor to VPO, VP3, and VP1 (Jore et al., 1988;
Ypma-Wong et al., 1988a). Cleavage of VPO to VP2 and VP4 is cata-
lyzed during or after RNA encapsidation and is widely believed to
occur intramolecularly (Arnold et al., 1987; Jacobson et al., 1970). The
viral proteins encoded in the P2 or P3 regions are released from poly-
protein precursors by the protease 3Cpro (Hanecak et al., 1982). These
cleavages occur'exclusively at glutamine-glycine dipeptides, although
6 DAVID C. ANSARDI et al.

not every glutamine-glycine dipeptide present in the genomic polypro-

tein is a substrate for 3C-mediated cleavages. An additional tyrosine-
glycine dipeptide substrate for 2A~rolies in the 3CD polyprotein, result-
ing in the production of two proteins, 3C' and 3D'. This cleavage may
simply be a fortuitous event as poliovirus mutants without this cleav-
age site have no apparent growth defects (Lee and Wimmer, 1988).

111. POLIOVIRUS
LIFECYCLE

Infection of cells by poliovirus is associated with several pronounced

cytopathic effects on the host cell, including shrinkage in cell size, an
increase in intracellular membranous vesicles, deformation of the nu-
cleus, and changes in the cell cytoskeleton (Koch and Koch, 1985). A
schematic representation of the events which take place during a sin-
gle cycle of poliovirus replication are depicted in Fig. 2. The virus
initially attaches to the host cell by binding to a cell-surface glyco-
protein molecule. The normal cellular function of the poliovirus recep-
tor is unknown, but the predicted amino acid sequence derived from
the cloning of the receptor gene indicates that the molecule belongs to
the immunoglobulin-like superfamily of proteins (Mendelsohn et al.,
1989). On attachment to the receptor, the virus undergoes conforma-
tional changes, and the internal capsid protein VP4 is expelled from
the virion (De La Torre et al., 1992; DeSena and Mandel, 1976, 1977;
Guttman and Baltimore, 1977a; Rueckert, 1990). The virus is believed
to be internalized into the cytoplasm by receptor-mediated endocytosis
(Madshus et al., 1984, 1985). The mechanism by which the virus re-
leases its RNA genome across the endosomal membrane and into the
cytoplasm is not understood.
Once present in the cytoplasm, the messenger-sense viral RNA ge-
nome is translated on host ribosomes to yield viral proteins. Transla-
tion of the poliovirus genome is an obligatory first step because the
virus does not package any of the proteins required to initiate replica-
tion of the viral RNA genome. An important consequence of poliovirus
infection is the shutoff of translation of host cell mRNA, which occurs
primarily as a result of cleavage of the large subunit ( ~ 2 2 0of
) the cap
binding complex (eIF-4F) (Etchison et al., 1982). This cleavage is indi-
rectly mediated by the viral protease 2Apro (Bernstein et al., 1985;
Krausslich et al., 1987; Lloyd et al., 1988; Wycoff et al., 1990). Once
viral proteins are synthesized, RNA synthesis occurs exponentially
from approximately 30 min postinfection to 3 hr postinfection, then
occurs in a linear fashion until approximately 4.5 hr postinfection
followed by a rapid decline in the rate of synthesis (Rueckert, 1990).
 POLIOVIRUS ASSEMBLY AND RNA ENCAPSIDATION 7

Replication of the viral genome requires that the plus-strand RNA

molecule is transcribed first to yield a complementary minus-strand
RNA, which is also linked at its 5' end to VPg (Kuhn and Wimmer,
1987; Paul et al., 1987b; Richards and Ehrenfeld, 1990). This minus-
strand RNA then serves as the template for synthesis of new plus
strands of RNA. Synthesis of plus- and minus-strand RNA molecules
is an asymmetric process, with plus strands produced in excess of
minus strands by at least 10-fold. Replication of the poliovirus RNA
genome occurs in association with smooth membrane vesicles which
proliferate on infection, and the combination of these membranes with
the viral proteins and RNA template molecules required for RNA rep-
lication is referred to as the replication complex (Caliguiri and Tamm,
1970; Ehrenfeld et al., 1970; Kuhn and Wimmer, 1987; Paul et al.,
1987b; Richards and Ehrenfeld, 1990). Progeny plus-strand RNA
serves as both mRNA for synthesis of additional viral proteins and as
the RNA molecule encapsidated in progeny virions. Encapsidated vir-
ion RNA is linked to VPg, whereas the VPg protein is removed from
the 5' end of plus-strand RNA molecules destined for translation
(Hewlett et al., 1976; Nomoto et al., 1977; Petterson et al., 1977).
The final aspect of the poliovirus life cycle is the formation of proge-
ny virions. The capsid proteins assemble subviral oligomeric particles,
probably prior to interaction with the RNA genome, although the pre-
cise pathway of assembly has not been deduced (Putnak and Phillips,
1981a; Rueckert, 1990).Encapsidation of plus-strand VPg-linked RNA
may occur by condensation of 12 pentamers of VPO, VP3, and VP1
[(VPO-3-1),] around the RNA molecule or by insertion of VPg-linked
RNA into a preformed empty capsid or procapsid consisting of 60 cop-
ies of VPO-VP3-VP1 [(VP0-3-1),,1 (Jacobson and Baltimore, 1968;
Rueckert, 1990). The encapsidation process is specific for both VPg-
linked RNA and plus strands as packaging of minus strands does not
occur, despite the presence of VPg (Nomoto et al., 1977; Novak and
Kirkegaard, 1991; Petterson et al., 1978). At the end of infection, lysis
of the cell occurs and virions exit, although mechanisms for active
release of virus prior to lysis may exist (Tucker et al., 1993).

A . Virus Entry and Uncoating

The mechanism by which poliovirus enters the host cell is poorly
understood (Rueckert, 1990). Progress in this field will likely proceed
at a faster pace with the identification, cloning, and sequencing of the
poliovirus receptor (Mendelsohn et al., 1989).
On attachment of poliovirus virions t o the glycoprotein receptor, the
virus undergoes conformational changes that are marked by a conver-
8 DAVID C. ANSARDI et al.

FIG.2. Events in a single cycle of poliovirus infection. Poliovirus virions initiate

infection by attaching to a glycoprotein receptor on the cell surface. The virus is believed
to be internalized into the cell by receptor-mediated endocytosis. On attachment to the
receptor and entry into the cell, the capsid undergoes conformational changes, and the
messenger-sense RNA genome is released into the cytoplasm in a n unknown manner.
The viral RNA genome is translated on host ribosomes to generate proteins required for
RNA replication and encapsidation of progeny genomes. RNA replication occurs in
virus-induced complexes of viral and host protein(s) that are associated with smooth
membrane vesicles. RNA replication proceeds by synthesis of minus-sense RNA fol-
lowed by synthesis of nascent plus strands, which occurs in excess over minus-strand
formation. RNA structures in which several nascent plus strands are simultaneously
being synthesized on the same minus-strand template are known as RI or replicative
 POLIOVIRUS ASSEMBLY AND RNA ENCAPSIDATION 9

sion of the sedimentation coefficient of the virion from 155s to 135s

(DeSena and Mandel, 1976, 1977; Everaert et al., 1989; Fricks and
Hogle, 1990; Guttman and Baltimore, 1977a;Kaplan et al., 1990).This
process is associated with the expulsion of the small, myristylated
internal capsid protein, VP4, from the virion. In addition to release of
VP4, the conformational changes associated with attachment to the
receptor also lead t o exposure of the amino terminus of the viral capsid
protein VP1 on the surface of the virion, a location which is far re-
moved from its normal location on the capsid interior (Fricks and
Hogle, 1990).Exposure of the amino terminus of VP1 on the surface of
the virion increases its hydrophobicity, giving the structurally altered
virions the ability to bind to liposomes (Fricks and Hogle, 1990).The
amino terminus of VP1 has been modeled as an amphipathic helix
(this region of VP1 was unresolved in the X-ray structure), and the
hypothetical formation of this structure has led to the proposal that
amino-terminal residues of VP1 may be involved in forming a pore
through endosomal membranes through which the viral RNA genome
can be released into the cytoplasm (Fricks and Hogle, 1990). A role
for the amino terminus of VP1 in virus uncoating has been supported
by the phenotypes of two temperature-sensitive poliovirus mutants
which contain small deletions in the VP1 amino terminus and which
are defective in virus uncoating at the nonpermissive temperature
(Kirkegaard, 1990; Kirkegaard and Nelson, 1990).
Attachment of virus to the cellular receptor is not a guarantee of
successful entry into the cell, as this process appears to be largely
abortive and is associated with sloughing of a large percentage of
attached, altered particles (Mandel, 1965; Rueckert, 1990).Once bound
to the receptor, the virus is believed to be internalized through
receptor-mediated endocytosis (Madshus et al., 1984, 1985). The pro-
cess by which RNA is released from the endosomes and into the cyto-
plasm is not well understood. Acidification of the endosomes might be
responsible for conformational changes required for capsid protein fu-
sion with the membrane and release of RNA (Madshus et al., 1984,
1985). A study conducted on a mutant of human rhinovirus, another
member of the Picornauiridae, suggested that the conformational
changes associated with receptor attachment were not sufficient for
RNA release into the cytoplasm, and led to the proposal that the un-
coating capsid must form a membrane-associated structure, termed an

intermediate RNA. Plus strands produced in the replication complexes are either encap-
sidated or translated (following removal of VPg) to generate additional viral proteins.
Poliovirus infection results in lysis of the host cell, allowing progeny virions to exit.
10 DAVID C. ANSARDI et al.

infectosome, responsible for injecting the RNA genome into the cyto-
plasm (Lee et al., 1993).

B . Translation of Viral RNA

Poliovirus has evolved a cap-independent method of translation
which allows it t o shut off host cell cap-dependent translation by inac-
tivating a component of a translation initiation factor (eIF-4F) which
recognizes the capped 5’ ends of host mRNA molecules (Etchison et al.,
1982; Pelletier et al., 1988; Pelletier and Sonenberg, 1988; Sonenberg,
1987, 1990; Trono et al., 1988). A host cellular enzyme is believed to
unlink the VPg protein from the 5’ end of poliovirus virion RNA prior
to translation (Ambros and Baltimore, 1978; Hewlett et al., 1976; Lee
et al., 1977; Morrow et al., 1984; Nomoto et al., 1977; Rothberg et al.,
1980; Wimmer, 1982).
Initiation of translation of poliovirus mRNA does not proceed by the
scanning model proposed by Kozak (1989). Ribosome binding to po-
liovirus RNA occurs in the 5’-NTR, upstream of the initiator AUG
codon, and is mediated by an internal sequence of several hundred
nucleotides, which has been designated the internal ribosome entry
sequence (IRES) (Jang et al., 1988; Pelletier et al., 1988; Pelletier and
Sonenberg, 1988; Sonenberg, 1990; Trono et al., 1988). The determi-
nants for recognition of the IRES by host translational machinery
have not been elucidated, but secondary RNA structures present in the
5’-NTR between nucleotides 240 and 620 may mediate the internal
binding of ribosomes (Sonenberg, 1990).How the IRES operates is not
yet clear; possibly the ribosome binds the IRES region and scans the
RNA genome until it encounters the initiator AUG codon at position
743 and begins translation. The 5‘-NTR of poliovirus type 1 contains
eight AUG codons upstream of the initiating AUG that are not used as
initiator codons (Kitamura et al., 1981; Racaniello and Baltimore,
1981a). However, the 100 nucleotides between the 3‘ end of the IRES
and the initiator methionine at nucleotide 743 contain no AUG codons.
Translation of the single long open reading frame results in the syn-
thesis of a long polyprotein. The actual existence of this translation
product in uiuo is doubtful, however, as 2Apro cleaves the P1 portion out
of the growing polyprotein intramolecularly and probably cotransla-
tionally (Toyoda et al., 1986).
The shutoff of host-cell mRNA translation in poliovirus-infected
cells is largely associated with cleavage of the p220 component of the
cap-binding complex (eIF-4F)(Etchison et al., 1982).Inactivation of the
cap-binding complex prevents binding of the translation initiation fac-
tors eIF-4A and eIF-4B to the 5’ end of mRNA molecules, which are