bluuebunny/crossref_metadata_2025 huggingface dataset:
The C Terminus of Brome Mosaic Virus Coat Protein Controls Viral Cell-to-Cell and Long-Distance Movement
Authors: Okinaka Yasushi , Mise Kazuyuki , Suzuki Eri , Okuno Tetsuro , Furusawa Iwao
Published: July 2002

To investigate the functional domains of the coat protein (CP; 189 amino acids) ofBrome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley andNicotiana benthamiana.Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.
Deletion Mapping of the Encephalomyocarditis Virus Primary Cleavage Site
Authors: Hahn Harry , Palmenberg Ann C.
Published: July 2002

The cotranslational, primary self-cleavage reaction of cardiovirus polyprotein relies on a highly conserved, short segment of amino acids at the 2A-2B protein boundary. The amino terminus of the required element for encephalomyocarditis virus has now been mapped to include Tyr
126
of the 2A protein, the 18th amino acid before the cleavage site.
Two Overlapping Subdominant Epitopes Identified by DNA Immunization Induce Protective CD8+T-Cell Populations with Differing Cytolytic Activities
Authors: Rodriguez Fernando , Slifka Mark K. , Harkins Stephanie , Whitton J. Lindsay
Published: July 2002

Subdominant CD8+T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by Ldand the other presented by Kd. This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8+T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8+response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the “dominant” vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the “subdominant” vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8+T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysislow-cytokinecompetentphenotype associated with most memory CD8+T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8+T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8+T cells with distinct functional attributes.
Tetramerization Is a Conserved Feature of the Virion-Associated Protein in Plant Pararetroviruses
Authors: Stavolone Livia , Herzog Etienne , Leclerc Denis , Hohn Thomas
Published: July 2002

All plant pararetroviruses belong to theCaulimoviridaefamily. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus ofCaulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.
Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase
Authors: Tai Chun-Ling , Pan Wen-Ching , Liaw Shwu-Huey , Yang Ueng-Cheng , Hwang Lih-Hwa , Chen Ding-Shinn
Published: July 2002

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.
Noncytolytic Inhibition of X4 Virus by Bulk CD8+Cells from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Persons and HIV-1-Specific Cytotoxic T Lymphocytes Is Not Mediated by β-Chemokines
Authors: Geiben-Lynn Ralf , Kursar Mischo , Brown Nancy V. , Kerr Ethan L. , Luster Andrew D. , Walker Bruce D.
Published: July 2002

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of β-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8+cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8+T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8+T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1α, MIP-1β, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8+cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.
Extensive Attenuation of Rabies Virus by Simultaneously Modifying the Dynein Light Chain Binding Site in the P Protein and Replacing Arg333 in the G Protein
Authors: Mebatsion Teshome
Published: July 2002

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/R
X
TQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein by an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.
Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation
Authors: Wang Shizhen , Liu Shuhong , Wu Ming-Hoi , Geng Yunqi , Wood Charles
Published: July 2002

Lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, from latency requires transcriptional transactivation by the viral protein RTA encoded by the ORF50 gene. Very little is known about how RTA functions and the cellular factors that may be involved in its transactivation function. Using the yeast two-hybrid system, we have identified a human cellular protein that can interact with KSHV RTA. The cellular protein, referred to as the human hypothetical protein MGC2663 by GenBank, is encoded by human chromosome 19. This protein is 554 amino acids (aa) in size and displays sequence similarity with members of the Krueppel-associated box–zinc finger proteins (KRAB-ZFPs). MGC2663 expression could be detected in all primate cell lines tested, and its expression level was neither stimulated nor inhibited by RTA. MGC2663 specifically synergizes with RTA to activate viral transcription, and overexpression of MGC2663 in the presence of RTA further enhances RTA transactivation of several viral promoters that were identified as targets for RTA. Coimmunoprecipitation and pull-down assays further demonstrated that MGC2663 interacts with RTA both in vivo and in vitro, and the N-terminal 273 aa of KSHV RTA and the potential zinc finger domain of MGC2663 are required for their interaction. Our results indicate that this novel human cellular protein, MGC2663, named K-RBP (KSHV RTA binding protein) due to its RTA binding feature, specifically interacts with the KSHV RTA protein and functions as a cellular RTA cofactor to activate viral gene expression. Though its normal cellular function needs to be further studied, K-RBP may play a significant role in mediating RTA transactivation in vivo.
Organization of Two Transmissible Gastroenteritis Coronavirus Membrane Protein Topologies within the Virion and Core
Authors: Escors David , Camafeita Emilio , Ortego Javier , Laude Hubert , Enjuanes Luis
Published: July 2002

The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of
35
S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.
Improved Elicitation of Neutralizing Antibodies against Primary Human Immunodeficiency Viruses by Soluble Stabilized Envelope Glycoprotein Trimers
Authors: Yang Xinzhen , Wyatt Richard , Sodroski Joseph
Published: July 2002

Human immunodeficiency virus (HIV-1) envelope glycoprotein subunits, such as the gp120 exterior glycoprotein, typically elicit antibodies that neutralize T-cell-line-adapted (TCLA), but not primary, clinical isolates of HIV-1. Here we compare the immunogenicity of gp120 and soluble stabilized trimers, which were designed to resemble the functional envelope glycoprotein oligomers of primary and TCLA HIV-1 strains. For both primary and TCLA virus proteins, soluble stabilized trimers generated neutralizing antibody responses more efficiently than gp120 did. Trimers derived from a primary isolate elicited antibodies that neutralized primary and TCLA HIV-1 strains. By contrast, trimers derived from a TCLA isolate generated antibodies that neutralized only the homologous TCLA virus. Thus, soluble stabilized envelope glycoprotein trimers derived from primary HIV-1 isolates represent defined immunogens capable of eliciting neutralizing antibodies that are active against clinically relevant HIV-1 strains.
Expression and Processing of Proteins Encoded by the
Saccharomyces
Retrotransposon Ty5
Authors: Irwin Phillip A. , Voytas Daniel F.
Published: July 2002

Retroelements (retrotransposons and retroviruses) have two genes in common:
gag
, which specifies structural proteins that form a virus or virus-like particle, and
pol
, which specifies catalytic proteins required for replication. For many retroelements,
gag
and
pol
are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The
Saccharomyces
retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after ∼14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.
Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage
Authors: Ross Heather L. , Nonnemacher Michael R. , Hogan Tricia H. , Quiterio Shane J. , Henderson Andrew , McAllister John J. , Krebs Fred C. , Wigdahl Brian
Published: July 2002

Recent observations have shown two CCAAT/enhancer binding protein (C/EBP) binding sites to be critically important for efficient human immunodeficiency virus type 1 (HIV-1) replication within cells of the monocyte/macrophage lineage, a cell type likely involved in transport of the virus to the brain. Additionally, sequence variation at C/EBP site I, which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB), has been shown to affect HIV-1 long terminal repeat (LTR) activity. Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family, we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I. Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent, naturally occurring, low-affinity C/EBP site. This biophysical interaction appears to occur via at least two mechanisms. First, low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites. In addition, CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites. This interaction is reciprocal, since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB. Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms. Most importantly, sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation, a treatment that leads to increases in C/EBP activation. Thus, HIV-1 LTR ATF/CREB binding site sequence variation may modulate cellular signaling at the viral promoter through the C/EBP pathway.
Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues
Authors: Bestman-Smith Julie , Schmit Isabelle , Papadopoulou Barbara , Boivin Guy
Published: July 2002

Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite
Leishmania
, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in
Leishmania
. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of
Leishmania
cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in
Leishmania
susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in
Leishmania
provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.
Imported Parakeets Harbor H9N2 Influenza A Viruses That Are Genetically Closely Related to Those Transmitted to Humans in Hong Kong
Authors: Mase Masaji , Imada Tadao , Sanada Yasuyuki , Etoh Mariko , Sanada Naoko , Tsukamoto Kenji , Kawaoka Yoshihiro , Yamaguchi Shigeo
Published: July 2002

In 1997 and 1998, H9N2 influenza A viruses were isolated from the respiratory organs of Indian ring-necked parakeets (
Psittacula Krameri manillensis
) that had been imported from Pakistan to Japan. The two isolates were closely related to each other (>99% as determined by nucleotide analysis of eight RNA segments), indicating that H9N2 viruses of the same lineage were maintained in these birds for at least 1 year. The hemagglutinins and neuraminidases of both isolates showed >97% nucleotide identity with those of H9N2 viruses isolated from humans in Hong Kong in 1999, while the six genes encoding internal proteins were >99% identical to the corresponding genes of H5N1 viruses recovered during the 1997 outbreak in Hong Kong. These results suggest that the H9N2 parakeet viruses originating in Pakistan share an immediate ancestor with the H9N2 human viruses. Thus, influenza A viruses with the potential to be transmitted directly to humans may be circulating in captive birds worldwide.
Sequences in the Cytoplasmic Tail of the Gibbon Ape Leukemia Virus Envelope Protein That Prevent Its Incorporation into Lentivirus Vectors
Authors: Christodoulopoulos Ilias , Cannon Paula M.
Published: July 2002

Pseudotyping retrovirus and lentivirus vectors with different viral fusion proteins is a useful strategy to alter the host range of the vectors. Although lentivirus vectors are efficiently pseudotyped by Env proteins from several different subtypes of murine leukemia virus (MuLV), the related protein from gibbon ape leukemia virus (GaLV) does not form functional pseudotypes. We have determined that this arises because of an inability of GaLV Env to be incorporated into lentivirus vector particles. By exploiting the homology between the GaLV and MuLV Env proteins, we have mapped the determinants of incompatibility in the GaLV Env. Three modifications that allowed GaLV Env to pseudotype human immunodeficiency virus type 1 particles were identified: removal of the R peptide (C-terminal half of the cytoplasmic domain), replacement of the whole cytoplasmic tail with the corresponding MuLV region, and mutation of two residues upstream of the R peptide cleavage site. In addition, we have previously proposed that removal of the R peptide from MuLV Env proteins enhances their fusogenicity by transmitting a conformational change to the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392–5398, 1998). Our analysis of chimeric MuLV/GaLV Env proteins provides further evidence in support of this model and suggests that proper Env function involves both interactions within the cytoplasmic tail and more long-range interactions between the cytoplasmic tail, the membrane-spanning region, and the ectodomain of the protein.
The E8 Domain Confers a Novel Long-Distance Transcriptional Repression Activity on the E8^E2C Protein of High-Risk Human Papillomavirus Type 31
Authors: Stubenrauch Frank , Zobel Thomas , Iftner Thomas
Published: July 2002

Infections with high-risk human papillomaviruses (HPVs) are the major risk factor for the development of anogenital cancers. Viral E2 proteins are involved in viral DNA replication and regulation of transcription. Repression of the viral P97 promoter by E2 proteins has been implicated in the modulation of the immortalization capacity and DNA replication properties of high-risk HPVs. Analysis of the
cis
and
trans
requirements for repression of the HPV type 31 (HPV31) P97 promoter, however, revealed striking differences between the full-length E2 and the E8^E2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8^E2C completely inhibited the P97 promoter from a single promoter-distal E2 binding site. This novel long-distance repression activity of the E8 domain also enabled E8^E2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8^E2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8^E2C might contribute to the progression of high-risk HPV-induced lesions.
Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag
Authors: Tobaly-Tapiero Joelle , Bittoun Patricia , Giron Marie-Lou , Neves Manuel , Koken Marcel , Saı̈b Ali , de Thé Hugues
Published: July 2002

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.
Infectious Virions Produced from a Human Papillomavirus Type 18/16 Genomic DNA Chimera
Authors: Meyers Craig , Bromberg-White Jennifer L. , Zhang Jiaping , Kaupas Michelle E. , Bryan Janine T. , Lowe Robert S. , Jansen Kathrin U.
Published: July 2002

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.
Mechanism of Virulence Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis Virus and Murray Valley Encephalitis Virus
Authors: Lee Eva , Lobigs Mario
Published: July 2002

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu
306
and Asp
390
for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.
Positive and Negative Modulation of Human Immunodeficiency Virus Type 1 Rev Function by
cis
and
trans
Regulators of Viral RNA Splicing
Authors: Pongoski Jodi , Asai Kengo , Cochrane Alan
Published: July 2002

Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. To investigate the possibility that modulation of viral RNA metabolism may alter Rev function, we analyzed the impact of multiple SR proteins on both processes. While overexpression of several of the SR factors altered splicing of HIV-1
env
mRNA, they had disparate effects on Rev function that varied with the cell line used. Subsequent examination of exon splicing enhancer (ESE) and/or silencer (ESS) deletions suggests that the effects of the SR proteins on Rev function are not mediated through interaction with these elements. However, analysis of the deletions did indicate that the ESE and/or ESS does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. In situ hybridization and reverse transcription-PCR indicated that the loss of Rev response upon deletion of the ESE was due to a failure of Rev to induce transport of the unspliced RNA into the cytoplasm. Together, the data indicate that cellular splicing factors and viral regulatory elements can have significant stimulatory and inhibitory effects on Rev function, raising the possibility that cells can be rendered permissive or nonpermissive for virus replication by modulation of splicing activities.
Blockade of Interferon Induction and Action by the E3L Double-Stranded RNA Binding Proteins of Vaccinia Virus
Authors: Xiang Ying , Condit Richard C. , Vijaysri Sangeetha , Jacobs Bertram , Williams Bryan R. G. , Silverman Robert H.
Published: July 2002

The vaccinia virus E3L gene encodes two double-stranded RNA binding proteins that promote viral growth and pathogenesis through suppression of innate immunity. To explore how E3L enables vaccinia virus to evade the interferon system, cells and mice deficient in the principal interferon-regulated antiviral enzymes, PKR and RNase L, were infected with wild-type vaccinia virus and strains of vaccinia virus from which E3L had been deleted (E3L-deleted strains). While wild-type virus was unaffected by RNase L and PKR, virus lacking E3L replicated only in the deficient cells. Nevertheless, E3L-deleted virus failed to replicate to high titers or to cause significant morbidity or mortality in triply deficient mice lacking RNase L, PKR, and Mx1. To investigate the underlying cause, we determined the effect of E3L on interferon regulatory factor 3 (IRF3), a transcription factor required for viral induction of subtypes of type I interferons. Results showed that IRF3 activation and interferon-β induction occurred after infections with E3L-deleted virus but not with wild-type virus. These findings demonstrate that E3L plays an essential role in the pathogenesis of vaccinia virus by blocking the interferon system at multiple levels. Furthermore, our results indicate the existence of an interferon-mediated antipoxvirus pathway that operates independently of PKR, Mx1, or the 2-5A/RNase L system.
Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10
Authors: Tibbles Lee Anne , Spurrell Jason C. L. , Bowen Gloria P. , Liu Qiang , Lam Mindy , Zaiss Anne K. , Robbins Stephen M. , Hollenberg Morley D. , Wickham Thomas J. , Muruve Daniel A.
Published: July 2002

The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMVβgal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMVβgal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or α
v
integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.
Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation
Authors: Takefman Daniel M. , Spear Gregory T. , Saifuddin Mohammed , Wilson Carolyn A.
Published: July 2002

Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.
Neurons Differentially Activate the Herpes Simplex Virus Type 1 Immediate-Early Gene ICP0 and ICP27 Promoters in Transgenic Mice
Authors: Loiacono Christie M. , Myers Robert , Mitchell William J.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the
Escherichia coli
β-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of β-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for β-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. β-Galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for β-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.
Budding of Equine Infectious Anemia Virus Is Insensitive to Proteasome Inhibitors
Authors: Patnaik Akash , Chau Vincent , Li Feng , Montelaro Ronald C. , Wills John W.
Published: July 2002

The only retrovirus protein required for the budding of virus-like particles is the Gag protein; however, recent studies of Rous sarcoma virus (RSV) and human immunodeficiency virus have suggested that modification of Gag with ubiquitin (Ub) is also required. As a consequence, the release of these viruses is reduced in the presence of proteasome inhibitors, which indirectly reduce the levels of free Ub within the cell. Here we show that the budding of equine infectious anemia virus (EIAV) from infected equine cells is largely unaffected by these drugs, although use of one inhibitor (MG-132) resulted in a dramatic block to proteolytic processing of Gag. This lack of sensitivity was also observed in transiently transfected avian cells under conditions that greatly reduce RSV budding. Moreover, insensitivity was observed when the EIAV Gag protein was expressed in the absence of all the other virus products, indicating that they are not required for this phenotype. An activity that enables EIAV to tolerate exposure to proteasome inhibitors was mapped to the C-terminal p9 sequence, as demonstrated by the ability of an RSV Gag-p9 chimera to bud in the presence of the drugs. Intriguingly, the p9 sequence contains a short sequence motif that is similar to a surface-exposed helix of Ub, suggesting that EIAV Gag may have captured a function that allows it to bypass the need for ubiquitination. Thus, the mechanism of EIAV budding may not be substantially different from that of other retroviruses, even though it behaves differently in the presence of proteasome inhibitors.
Novel, Chimpanzee Serotype 68-Based Adenoviral Vaccine Carrier for Induction of Antibodies to a Transgene Product
Authors: Xiang Zhiquan , Gao Guangping , Reyes-Sandoval Arturo , Cohen Christopher J. , Li Yan , Bergelson Jeffrey M. , Wilson James M. , Ertl Hildegund C. J.
Published: July 2002

An E1-deletion-containing adenoviral recombinant based on the chimpanzee serotype 68 (AdC68) was developed to express the rabies virus glycoprotein. Mice immunized with this construct (AdC68rab.gp) developed antibodies to rabies virus and remained resistant to challenge with an otherwise lethal dose of rabies virus. In naïve mice immunized intranasally, the rabies virus-specific antibody responses elicited by AdC68rab.gp were comparable with regard to both titers and isotype profiles to those induced by an adenoviral recombinant based on human serotype 5 (Adhu5) expressing the same transgene product. In contrast, subcutaneous immunization with the AdC68rab.gp vaccine resulted in markedly lower antibody responses to the rabies virus glycoprotein than the corresponding Adhu5 vaccine. Antibodies from AdC68rab.gp-immunized mice were strongly biased towards the immunoglobulin G2a isotype. The antibody response to the rabies virus glycoprotein presented by Adhu5rab.gp was severely compromised in animals preexposed to the homologous adenovirus. In contrast, the rabies virus-specific antibody response to the AdC68rab.gp vaccine was at most marginally affected by preexisting immunity to common human adenovirus serotypes, such as 2, 4, 5, 7, and 12. This novel vaccine carrier thus offers a distinct advantage over adenoviral vaccines based on common human serotypes.
Improving DNA Vaccine Potency by Linking Marek's Disease Virus Type 1 VP22 to an Antigen
Authors: Hung Chien-Fu , He Liangmei , Juang Jeremy , Lin Tzyy-Jye , Ling Morris , Wu T.-C.
Published: July 2002

We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Marek's disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8+-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency.
Novel Swine Virulence Determinant in the Left Variable Region of the African Swine Fever Virus Genome
Authors: Neilan J. G. , Zsak L. , Lu Z. , Kutish G. F. , Afonso C. L. , Rock D. L.
Published: July 2002

Previously we have shown that the African swine fever virus (ASFV)
NL
gene deletion mutant E70ΔNL is attenuated in pigs. Our recent observations that
NL
gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70
NL
deletion mutant virus (E70ΔNL) and cosmid DNA clones from the Malawi
NL
gene deletion mutant (MalΔNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalΔNL genome was capable of restoring full virulence to E70ΔNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70ΔNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalΔNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70ΔNL. Comparative nucleotide sequence analysis of the left variable region of the E70ΔNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70ΔNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalΔNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.