bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Binds to p53 and Represses p53-Dependent Transcription and Apoptosis
Authors: Seo Taegun , Park Junsoo , Lee Daeyoup , Hwang Sun Gwan , Choe Joonho
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) is related to the development of Kaposi's sarcoma. Open reading frame K9 of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon- and IRF1-mediated signal transduction. In addition, vIRF1 acts as an oncogene to induce cellular transformation. Here we show that vIRF1 directly associates with the tumor suppressor p53 and represses its functions. The vIRF1 interaction domains of p53 are the DNA binding domain (amino acids [aa] 100 to 300) and the tetramerization domain (aa 300 to 393). p53 interacts with the central region (aa 152 to 360) of vIRF1. vIRF1 suppresses p53-dependent transcription and deregulates its apoptotic activity. These results suggest that vIRF1 may regulate cellular function by inhibiting p53.
Variant Chromatin Structure of the
oriP
Region of Epstein-Barr Virus and Regulation of EBER1 Expression by Upstream Sequences and
oriP
Authors: Wensing Barbara , Stühler Albert , Jenkins Peter , Hollyoake Martine , Karstegl Claudio Elgueta , Farrell Paul J.
Published: July 2002

Most of the Epstein-Barr virus genome in latently infected cells is in a standard nucleosomal structure, but the region encompassing
oriP
and the Epstein-Barr virus-encoded small RNA (EBER) genes shows a distinctive pattern when digested with micrococcal nuclease. This pattern corresponds to a previously mapped nuclear matrix attachment region. Although the EBER genes are adjacent to
oriP
, there is only a two- to fourfold effect of
oriP
on EBER expression. However, sequences containing a consensus ATF site upstream of EBER1 are important for EBER1 expression.
Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2
Authors: Alcorn M. J. , Booth J. L. , Coggeshall K. M. , Metcalf J. P.
Published: July 2002

Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.
Cell-Type-Specific Regulation of the Two Foamy Virus Promoters
Authors: Meiering Christopher D. , Rubio Claudia , May Cynthia , Linial Maxine L.
Published: July 2002

The foamy virus (FV) genome contains two promoters, the canonical long terminal repeat (LTR) promoter, containing three consensus AP-1 binding sites, and an internal promoter (IP) within theenvgene. We investigated the regulation of the two promoters in lytic and persistent infections and found that in the presence of a constitutive source of the viral transactivator protein Tas, transactivation of the LTR promoter and that of the IP differ. In lytic infections, both the LTR promoter and the IP are efficiently transactivated by Tas, while in persistent infections, the IP is efficiently transactivated by Tas, but the LTR promoter is not. Analysis of proteins expressed from the LTR promoter and the IP during infection indicated that IP transcription is more robust than that of the LTR promoter in persistently infected cells, while the opposite is true for lytically infected cells. Coculture experiments also showed that LTR promoter transcription is greatest in cells which support lytic replication. Replacement of much of the LTR promoter with the IP leads to increased viral replication in persistent but not lytic infections. We also found that the induction of persistently infected cells with phorbol 12-myristate 13-acetate (PMA) greatly enhanced viral replication and transcription from the SFVcpz(hu) (new name for human FV) LTR promoter. However, mutation of three consensus AP-1 binding sites in the FV LTR promoter did not affect viral replication in lytically or persistently infected cells, nor did the same mutations affect LTR promoter transactivation by Tas in PMA-treated cells. Our data indicate that differential regulation of transcription is important in the outcome of FV infection but is unlikely to depend on AP-1.
Antibody from Patients with Acute Human Immunodeficiency Virus (HIV) Infection Inhibits Primary Strains of HIV Type 1 in the Presence of Natural-Killer Effector Cells
Authors: Forthal Donald N. , Landucci Gary , Daar Eric S.
Published: July 2002

The partial control of viremia during acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by an HIV-1-specific cytotoxic T-lymphocyte (CTL) response and an absent or infrequent neutralizing antibody response. The control of HIV-1 viremia has thus been attributed primarily, if not exclusively, to CTL activity. In this study, the role of antibody in controlling viremia was investigated by measuring the ability of plasma or immunoglobulin G from acutely infected patients to inhibit primary strains of HIV-1 in the presence of natural-killer (NK) effector cells. Antibody that inhibits virus when combined with effector cells was present in the majority of patients within days or weeks after onset of symptoms of acute infection. Furthermore, the magnitude of this effector cell-mediated antiviral antibody response was inversely associated with plasma viremia level, and both autologous and heterologous HIV-1 strains were inhibited. Finally, antibody from acutely infected patients likely reduced HIV-1 yield in vitro both by mediating effector cell lysis of target cells expressing HIV-1 glycoproteins and by augmenting the release of β-chemokines from NK cells. HIV-1-specific antibody may be an important contributor to the early control of HIV viremia.
N-Terminal Domain of Borna Disease Virus G (p56) Protein Is Sufficient for Virus Receptor Recognition and Cell Entry
Authors: Perez Mar , Watanabe Michiko , Whitt Michael A. , de la Torre Juan Carlos
Published: July 2002

Borna disease virus (BDV) surface glycoprotein (GP) (p56) has a predicted molecular mass of 56 kDa. Due to extensive posttranslational glycosylation the protein migrates as a polypeptide of 84 kDa (gp84). The processing of gp84 by the cellular protease furin generates gp43, which corresponds to the C-terminal part of gp84. Both gp84 and gp43 have been implicated in viral entry involving receptor-mediated endocytosis and pH-dependent fusion. We have investigated the domains of BDV p56 involved in virus entry. For this, we used a pseudotype approach based on a recently developed recombinant vesicular stomatitis virus (VSV) in which the gene for green fluorescent protein was substituted for the VSV G protein gene (VSVΔG*). Complementation of VSVΔG* with BDV p56 resulted in infectious VSVΔG* pseudotypes that contained both BDV gp84 and gp43. BDV-VSV chimeric GPs that contained the N-terminal 244 amino acids of BDV p56 and amino acids 421 to 511 of VSV G protein were efficiently incorporated into VSVΔG* particles, and the resulting pseudotype virions were neutralized by BDV-specific antiserum. These findings indicate that the N-terminal part of BDV p56 is sufficient for receptor recognition and virus entry.
A Virally Encoded Chaperone Specialized for Folding of the Major Capsid Protein of African Swine Fever Virus
Authors: Cobbold C. , Windsor M. , Wileman T.
Published: July 2002

It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of
African swine fever virus
(ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.
Inhibition of p53 Tumor Suppressor by Viral Interferon Regulatory Factor
Authors: Nakamura Hiroyuki , Li Mengtao , Zarycki Jodi , Jung Jae U.
Published: July 2002

The irreversible cell cycle arrest and apoptosis induced by p53 are part of the host surveillance mechanisms for viral infection and tumor induction. Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumor virus, is associated with the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The K9 open reading frame of KSHV encodes a viral interferon (IFN) regulatory factor (vIRF) which functions as a repressor for cellular IFN-mediated signal transduction and as an oncoprotein to induce cell growth transformation. Here, we demonstrate that KSHV vIRF interacts with the cellular p53 tumor suppressor through the putative DNA binding region of vIRF and the central region of p53. This interaction suppresses the level of phosphorylation and acetylation of p53 and inhibits transcriptional activation of p53. As a consequence, vIRF efficiently prevents p53-mediated apoptosis. These results suggest that KSHV vIRF interacts with and inhibits the p53 tumor suppressor to circumvent host growth surveillance and to facilitate uncontrolled cell proliferation.
Early Polyadenylation Signals of Human Papillomavirus Type 31 Negatively Regulate Capsid Gene Expression
Authors: Terhune Scott S. , Hubert Walter G. , Thomas Jennifer T. , Laimins Laimonis A.
Published: July 2002

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5′-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.
Concerted Action of the FasL/Fas and Perforin/Granzyme A and B Pathways Is Mandatory for the Development of Early Viral Hepatitis but Not for Recovery from Viral Infection
Authors: Balkow Sandra , Kersten Astrid , Tran Thi Thanh Thao , Stehle Thomas , Grosse Philipp , Museteanu Crisan , Utermöhlen Olaf , Pircher Hanspeter , von Weizsäcker Fritz , Wallich Reinhard , Müllbacher Arno , Simon Markus M.
Published: July 2002

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.
Study of the Mechanism of Antiviral Action of Iminosugar Derivatives against Bovine Viral Diarrhea Virus
Authors: Durantel David , Branza-Nichita Norica , Carrouée-Durantel Sandra , Butters Terry D. , Dwek Raymond A. , Zitzmann Nicole
Published: July 2002

The glucose-derived iminosugar derivatives
N
-butyl- and
N
-nonyl-deoxynojirimycin (DNJ) have an antiviral effect against a broad spectrum of viruses including
Bovine viral diarrhea virus
(BVDV). For BVDV, this effect has been attributed to the reduction of viral secretion due to an impairment of viral morphogenesis caused by the ability of DNJ-based iminosugar derivatives to inhibit ER α-glucosidases (N. Zitzmann, A. S. Mehta, S. Carrouée, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, and T. M. Block, Proc. Natl. Acad. Sci. USA 96:11878–11882, 1999). Here we present the antiviral features of newly designed DNJ derivatives and report for the first time the antiviral activity of long-alkyl-chain derivatives of deoxygalactonojirimycin (DGJ), a class of iminosugars derived from galactose which does not inhibit endoplasmic reticulum (ER) α-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER α-glucosidases and their antiviral effect, ruling out ER α-glucosidase inhibition as the sole mechanism responsible. Using short- and long-alkyl-chain DNJ and DGJ derivatives, we investigated the mechanisms of action of these drugs. First, we excluded their potential action at the level of the replication, protein synthesis, and protein processing. Second, we demonstrated that DNJ derivatives cause both a reduction in viral secretion and a reduction in the infectivity of newly released viral particles. Long-alkyl-chain DGJ derivatives exert their antiviral effect solely via the production of viral particles with reduced infectivity. We demonstrate that long-alkyl-chain DNJ and DGJ derivatives induce an increase in the quantity of E2-E2 dimers accumulated within the ER. The subsequent enrichment of these homodimers in secreted virus particles correlates with their reduced infectivity.
Viral Evolution toward Change in Receptor Usage: Adaptation of a Major Group Human Rhinovirus To Grow in ICAM-1-Negative Cells
Authors: Reischl Andrea , Reithmayer Manuela , Winsauer Gabriele , Moser Rosita , Gösler Irene , Blaas Dieter
Published: July 2002

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a “catalytic” function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.
In Vivo Selection of T-Cell Receptor Junctional Region Sequences by HLA-A2 Human T-Cell Lymphotropic Virus Type 1 Tax11-19 Peptide Complexes
Authors: Saito Mineki , Taylor Graham P. , Saito Akiko , Furukawa Yoshitaka , Usuku Koichiro , Weber Jonathan N. , Osame Mitsuhiro , Bangham Charles R. M.
Published: July 2002

Using HLA-peptide tetrameric complexes, we isolated human T-cell lymphotrophic virus type 1 Tax peptide-specific CD8+T cells ex vivo. Antigen-specific amino acid motifs were identified in the T-cell receptor Vβ CDR3 region of clonally expanded CD8+T cells. This result directly confirms the importance of the CDR3 region in determining the antigen specificity in vivo.
Vaccination with aShigellaDNA Vaccine Vector Induces Antigen-Specific CD8+T Cells and Antiviral Protective Immunity
Authors: Shata Mohamed T. , Hone David M.
Published: July 2002

A prototypeShigellahuman immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env(vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8+-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of theShigellaHIV-1 gp120 DNA vaccine vector elicited a CD8+T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-envvector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of theShigellagp120 DNA vaccine vector afforded significant protection against a vaccinia-envchallenge. Moreover, the number of vaccinia-envPFU recovered in mice vaccinated intranasally with theShigellavector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since theShigellavector did not express detectable levels of gp120, this report confirms thatShigellavectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8+T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a liveShigellaDNA vaccine vector.
Multiple Effector Functions Mediated by Human Immunodeficiency Virus-Specific CD4+T-Cell Clones
Authors: Norris Philip J. , Sumaroka Marina , Brander Christian , Moffett Howell F. , Boswell Steven L. , Nguyen Tam , Sykulev Yuri , Walker Bruce D. , Rosenberg Eric S.
Published: July 2002

Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4+T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-γ) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.
Rhabdoviruses and the Cellular Ubiquitin-Proteasome System: a Budding Interaction
Authors: Harty Ronald N. , Brown Melissa E. , McGettigan James P. , Wang Guangli , Jayakar Himangi R. , Huibregtse Jon M. , Whitt Michael A. , Schnell Matthias J.
Published: July 2002

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.
Selective Loss of Natural Killer T Cells by Apoptosis following Infection with Lymphocytic Choriomeningitis Virus
Authors: Hobbs Jacqueline A. , Cho Sungyoo , Roberts Tonya J. , Sriram Venkataraman , Zhang Jianhua , Xu Ming , Brutkiewicz Randy R.
Published: July 2002

Natural killer T (NKT) cells, a unique subpopulation of T cells, coexpress markers also present on NK cells and recognize the major histocompatibility complex class I-like CD1d1 molecule. We studied the effect of an acute virus infection on NKT cells. Mice were infected with the nonhepatotropic Armstrong strain of lymphocytic choriomeningitis virus (LCMV), and at various times postinfection, mononuclear cells from the liver, peritoneum, and spleen were isolated. It was found that within 2 to 3 days, there was a selective loss of NKT cells from the liver with an apparent rapid recovery within 8 to 14 days. There was no increase in peritoneal or splenic NKT cells, indicating that NKT cells did not traffic to these tissues. This loss of NKT cells was independent of gamma interferon (IFN-γ) and interleukin 12 (IL-12) production, but did occur in mice treated with poly(I-C), a classical inducer of IFN-α/β. The reduction in NKT cells was CD28 andfas/fasLindependent and occurred via apoptosis. It was not observed in LCMV-infected DNA fragmentation factor 45-deficient mice, and an increase in active caspase 3-specific staining was found in liver NKT cells from LCMV-infected and poly(I-C)-treated mice compared to uninfected wild-type mice. Interestingly, it was also found that liver NKT cells from LCMV-infected mice were themselves infected. These results suggest that the loss of NKT cells following an acute LCMV infection could be due to the induction of IFN-α/β resulting in NKT-cell apoptosis and is important for the host's immune response to LCMV.
Packaging of Genomic and Amplicon DNA by the Herpes Simplex Virus Type 1 UL25-Null Mutant KUL25NS
Authors: Stow Nigel D.
Published: July 2002

The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060–1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into the cytoplasm.
Role of Polypyrimidine Tract Binding Protein in the Function of the Hepatitis B Virus Posttranscriptional Regulatory Element
Authors: Zang Wei-Qing , Li Bin , Huang Pei-Yong , Lai Michael M. C. , Yen T. S. Benedict
Published: July 2002

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.
Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion
Authors: Finnegan Catherine M. , Berg Werner , Lewis George K. , DeVico Anthony L.
Published: July 2002

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV
HXB2
envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4
°
C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.
Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis
Authors: Lu Min , Stoller Marisa O. , Wang Shilong , Liu Jie , Fagan Melinda B. , Nunberg Jack H.
Published: July 2002

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.
Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission
Authors: Mikloska Zorka , Cunningham Anthony L.
Published: July 2002

The ability of alpha interferon (IFN-α) and IFN-γ to inhibit transmission of herpes simplex virus type 1 (HSV-1) from neuronal axon to epidermal cells (ECs), and subsequent spread in these cells was investigated in an in vitro dual-chamber model consisting of human fetal dorsal root ganglia (DRG) innervating autologous skin explants and compared with direct HSV-1 infection of epidermal explants. After axonal transmission from HSV-1-infected DRG neurons, both the number and size of viral cytopathic plaques in ECs was significantly reduced by addition of recombinant IFN-γ and IFN-α to ECs in the outer chamber in a concentration-dependent fashion. Inhibition was maximal when IFNs were added at the same time as the DRG were infected with HSV-1. The mean numbers of plaques were reduced by 52% by IFN-α, 36% by IFN-γ, and by 62% when IFN-α and IFN-γ were combined, and the mean plaque size was reduced by 64, 43, and 72%, respectively. Similar but less-inhibitory effects of both IFNs were observed after direct infection of EC explants, being maximal when IFNs were added simultaneously or 6 h before HSV-1 infection. These results show that both IFN-α and IFN-γ can interfere with HSV-1 infection after axonal transmission and subsequent spread of HSV-1 in ECs by a direct antiviral effect. Therefore, both IFN-α and -γ could contribute to the control of HSV-1 spread and shedding in a similar fashion in recurrent herpetic lesions.
Mechanisms Governing Expression of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Grundhoff Adam , Ganem Don
Published: July 2002

Open reading frame 71 (ORF 71) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a death effector domain-containing protein that is homologous to cellular FLIPs (FLICE-inhibitory proteins) and is proposed to inhibit Fas-mediated apoptosis. Transcripts bearing ORF 71 (v-FLIP) sequences are present in all latently infected cells. However, mapping studies reveal these to be bi- or tricistronic mRNAs with ORF 71 located 3′ to ORFs 72 (v-cyclin) and 73 (latency-associated nuclear antigen), raising the question of how efficient expression of v-FLIP is achieved. We explored this question by examining the expression of model bicistronic (v-cyclin/LUC) transcripts in which a luciferase (LUC) reporter replaced v-FLIP coding sequences. SLK spindle cells transfected with such constructs efficiently expressed luciferase from the 3′ position, and this expression was independent of the expression of the 5′ v-cyclin gene. Surprisingly, transcript mapping showed that in these cultures, efficient splicing occurred to remove v-cyclin sequences and generate monocistronic LUC transcripts. Similar splicing events produced monocistronic v-FLIP transcripts in KSHV-infected primary effusion lymphoma cells. However, these RNAs were of low abundance and were inducible by treatment with 12-
O
-tetradecanoylphorbol-13-acetate. Examination of the more abundant bicistronic latent RNAs revealed the presence of an efficient internal ribosome entry site (IRES) overlapping ORF 72 coding sequences. Thus, two potential mechanisms exist for v-FLIP expression, but the evidence suggests that IRES-mediated internal translational initiation on latent polycistronic mRNAs is the principal source of v-FLIP in latency.
Cytoplasmic Domain of Herpes Simplex Virus gE Causes Accumulation in the
trans
-Golgi Network, a Site of Virus Envelopment and Sorting of Virions to Cell Junctions
Authors: McMillan Tom N. , Johnson David C.
Published: July 2002

Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to the
trans
-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gI
CT
chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gE
CT
chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gE
CT
, gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI
−
HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004–4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821–833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.
cis
-Acting Signals in Encapsidation of Hantaan Virus S-Segment Viral Genomic RNA by Its N Protein
Authors: Severson William E. , Xu Xiaolin , Jonsson Colleen B.
Published: July 2002

The nucleocapsid (N) protein encapsidates both viral genomic RNA (vRNA) and the antigenomic RNA (cRNA), but not viral mRNA. Previous work has shown that the N protein has preference for vRNA, and this suggested the possibility of a
cis
-acting signal that could be used to initiate encapsidation for the S segment. To map the
cis
-acting determinants, several deletion RNA derivatives and synthetic oligoribonucleotides were constructed from the S segment of the Hantaan virus (HTNV) vRNA. N protein-RNA interactions were examined by UV cross-linking studies, filter-binding assays, and gel electrophoresis mobility shift assays to define the ability of each to bind HTNV N protein. The 5′ end of the S-segment vRNA was observed to be necessary and sufficient for the binding reaction. Modeling of the 5′ end of the vRNA revealed a possible stem-loop structure (SL) with a large single-stranded loop. We suggest that a specific interaction occurs between the N protein and sequences within this region to initiate encapsidation of the vRNAs.
Ross River Virus Glycoprotein-Pseudotyped Retroviruses and Stable Cell Lines for Their Production
Authors: Sharkey C. Matthew , North Cynthia L. , Kuhn Richard J. , Sanders David Avram
Published: July 2002

Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.
Mouse-Human Heterokaryons Support Efficient Human Immunodeficiency Virus Type 1 Assembly
Authors: Mariani Roberto , Rasala Beth A. , Rutter Gabriel , Wiegers Klaus , Brandt Stephanie M. , Kräusslich Hans-Georg , Landau Nathaniel R.
Published: July 2002

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication.Mus musculuslymphoid cell lines EL4 and L1-2 andMus dunnifibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infectedM. dunnitail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.
Borna Disease Virus Nucleoprotein Requires both Nuclear Localization and Export Activities for Viral Nucleocytoplasmic Shuttling
Authors: Kobayashi Takeshi , Kamitani Wataru , Zhang Guoqi , Watanabe Makiko , Tomonaga Keizo , Ikuta Kazuyoshi
Published: July 2002

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order
Mononegavirales
and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.
Replication of the Human Hepatitis Delta Virus Genome Is Initiated in Mouse Hepatocytes following Intravenous Injection of Naked DNA or RNA Sequences
Authors: Chang Jinhong , Sigal Luis J. , Lerro Anthony , Taylor John
Published: July 2002

As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even
in vitro
-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258–1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.