bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Controlled Incorporation of Herpes Simplex Virus Type 1 UL26 Protease into Capsids
Authors: Sheaffer Amy K. , Newcomb William W. , Brown Jay C. , Gao Min , Weller Sandra K. , Tenney Daniel J.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) capsids are initially assembled with an internal protein scaffold. The scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain, and its C-terminal oligomerization and capsid protein-binding domains are identical to those of UL26.5. The UL26 protease cleaves itself, releasing minor scaffold proteins VP24 and VP21, and the more abundant UL26.5 protein, releasing the major scaffold protein VP22a. Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 (containing the protease domain) is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention, we used a mutant virus that fails to express UL26.5 (Δ
ICP35
virus). Purified Δ
ICP35
B capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B capsids but contained capsid shell proteins in wild-type amounts. Despite C-terminal sequence identity between UL26 and UL26.5, Δ
ICP35
capsids lacking UL26.5 products did not contain compensatory high levels of UL26 proteins. Therefore, HSV capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not compensate for the Δ
ICP35
growth defect. While indirect, these findings are consistent with the view that UL26 products are restricted from occupying abundant UL26.5 binding sites within the capsid and that this restriction is not controlled by the level of UL26 protein expression. Additionally, Δ
ICP35
capsids contained an altered complement of DNA cleavage and packaging proteins, suggesting a previously unrecognized role for the scaffold in this process.
Induction of p53-Independent Apoptosis by the Adenovirus E4orf4 Protein Requires Binding to the Bα Subunit of Protein Phosphatase 2A
Authors: Marcellus Richard C. , Chan Helen , Paquette Denis , Thirlwell Sarah , Boivin Dominique , Branton Philip E.
Published: July 2002

Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Bα subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Bα-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Bα subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing.
The Genome of a Very Virulent Marek's Disease Virus
Authors: Tulman E. R. , Afonso C. L. , Lu Z. , Zsak L. , Rock D. L. , Kutish G. F.
Published: July 2002

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.
γδ+T-Lymphocyte Cytotoxicity against Envelope-Expressing Target Cells Is Unique to the Alymphocytic State of Bovine Leukemia Virus Infection in the Natural Host
Authors: Lundberg Patric , Splitter Gary A.
Published: July 2002

Bovine leukemia virus (BLV) is a complex B-lymphotrophic retrovirus of cattle and the causative agent of enzootic bovine leukosis. Serum antibody in infected animals does not correlate with protection from disease, yet only some animals develop severe disease. While a cytotoxic T-lymphocyte response may be responsible for directing BLV pathogenesis, this possibility has been left largely unexplored, in part since the lack of readily established cytotoxic target cells in cattle has hampered such studies. Using long-term naturally infected alymphocytic (AL) cattle, we have established the existence of cytotoxic T-lymphocyte response against BLV envelope proteins (Env; gp51/gp30). In vitro-expanded peripheral blood mononuclear (PBM) cell effector populations consisted mainly of γδ+(>40%), CD4+(>35%), and CD8+(>10%) T lymphocytes. Specific lysis of autologous fibroblasts infected with recombinant vaccinia virus (rVV) delivering the BLVenvgene ranged from 30 to 65%. Depletion studies indicated that γδ+and not CD8+T cells were responsible for the cytotoxicity against autologous rVVenv-expressing fibroblasts. Additionally, cultured effector cells lysed rVVenv-expressing autologous fibroblasts and rVVenv-expressing xenogeneic targets similarly, suggesting a lack of genetic restricted killing. Restimulation of effector populations increased the proportion of γδ+T cells and concomitantly Env-specific cytolysis. Interestingly, culture of cells from BLV-negative or persistently lymphocytic cattle failed to elicit such cytotoxic responses or increase in γδ+T-cell numbers. These results imply that cytotoxic γδ+T lymphocytes from only AL cattle recognize BLV Env without a requirement for classical major histocompatibility complex interactions. It is known that γδ+T lymphocytes are diverse and numerous in cattle, and here we show that they may serve a surveillance role during natural BLV infection.
Retracing the Evolutionary Pathways of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors: Virus Fitness in the Absence and in the Presence of Drug
Authors: Mammano Fabrizio , Trouplin Virginie , Zennou Veronique , Clavel Francois
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) is a major obstacle to the full success of combined antiretroviral therapy. High-level resistance to these compounds is the consequence of stepwise accumulation of amino acid substitutions in the HIV-1 protease (PR), following pathways that usually differ from one inhibitor to another. The selective advantage conferred by resistance mutations may depend upon several parameters: the impact of the mutation on virus infectivity in the presence or absence of drug, the nature of the drug, and its local concentration. Because drug concentrations in vivo are subject to extensive variation over time and display a markedly uneven tissue distribution, the parameters of selection for HIV-1 resistance to PI in treated patients are complex and poorly understood. In this study, we have reconstructed a large series of HIV-1 mutants that carry single or combined mutations in the PR, retracing the accumulation pathways observed in ritonavir-, indinavir-, and saquinavir-treated patients. We have then measured the phenotypic resistance and the drug-free infectivity of these mutant viruses. A deeper insight into the evolutionary value of HIV-1 PR mutants came from a novel assay system designed to measure the replicative advantage of mutant viruses as a function of drug concentration. By tracing the resultant fitness profiles, we determined the range of drug concentrations for which mutant viruses displayed a replicative advantage over the wild type and the extent of this advantage. Fitness profiles were fully consistent with the order of accumulation of resistance mutations observed in treated patients and further emphasise the key importance of local drug concentration in the patterns of selection of drug-resistant HIV-1 mutants.
Transcriptional Regulation of the Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor Gene
Authors: Chen Jiguo , Ueda Keiji , Sakakibara Shuhei , Okuno Toshiomi , Yamanishi Koichi
Published: July 2002

The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5′ rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate β-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.
Protein Synthesis Shut-Off Induced by Influenza Virus Infection Is Independent of PKR Activity
Authors: Zürcher Thomas , Marión Rosa María , Ortín Juan
Published: July 2002

The role of PKR activity in influenza virus-induced cell shut-off was studied by infection of PKR
+
or PKR
−
cell cultures and metabolic labeling in vivo. No differences in the synthesis of viral proteins or the decay of cellular protein synthesis were observed. To investigate the relevance of the inhibition of cellular pre-mRNA polyadenylation and nucleocytoplasmic transport in virus-induced shut-off, we carried out similar experiments with mutant viruses lacking C-terminal sequences of NS1 protein. No differences in the shut-off induced by mutant versus wild-type viruses were observed, indicating that these nuclear events are not relevant for shut-off. The analysis of cytoplasmic mRNA stability indicated that the accumulation of viral mRNA during the infection correlated with the progressive decay of cellular mRNA, in both the wild type and an NS1 deletion mutant.
Attenuation of Tick-Borne Encephalitis Virus by Structure-Based Site-Specific Mutagenesis of a Putative Flavivirus Receptor Binding Site
Authors: Mandl Christian W. , Allison Steven L. , Holzmann Heidemarie , Meixner Tamara , Heinz Franz X.
Published: July 2002

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3′-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.
Porcine Reproductive and Respiratory Syndrome Virus: Description of Persistence in Individual Pigs upon Experimental Infection
Authors: Allende R. , Laegreid W. W. , Kutish G. F. , Galeota J. A. , Wills R. W. , Osorio F. A.
Published: July 2002

We studied the persistence of porcine reproductive and respiratory syndrome virus (PRRSV) in individual experimentally infected pigs, during a period of up to 150 days postinfection (dpi). The results of this study suggest that the persistence of PRRSV involves continuous viral replication but that it is not a true steady-state persistent infection. The virus eventually clears the body and seems to do it in most of the animals by 150 dpi or shortly thereafter. High genetic stability was seen for several regions of the persistent PRRSV's genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.
A Baculovirus Superinfection System: Efficient Vehicle for Gene Transfer into
Drosophila
S2 Cells
Authors: Lee Dung-Fang , Chen Chun-Chen , Hsu Tsu-An , Juang Jyh-Lyh
Published: July 2002

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood
Drosophila
S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a
Drosophila
heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized
Drosophila
S2 cells.
The In Vitro-Synthesized RNA from a cDNA Clone of Hepatitis E Virus Is Infectious
Authors: Panda S. K. , Ansari I. H. , Durgapal H. , Agrawal S. , Jameel S.
Published: July 2002

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [
35
S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (
Macaca mulatta
) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.
Covalent Modification of the Transactivator Protein IE2-p86 of Human Cytomegalovirus by Conjugation to the Ubiquitin-Homologous Proteins SUMO-1 and hSMT3b
Authors: Hofmann Heike , Flöss Stefan , Stamminger Thomas
Published: July 2002

The 86-kDa IE2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a potent transactivator of viral as well as cellular promoters. Several lines of evidence indicate that this broad transactivation spectrum is mediated by protein-protein interactions. To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the isolation of two ubiquitin-homologous proteins, SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2. The polypeptides SUMO-1 and hSMT3b have previously been shown to be covalently coupled to a subset of nuclear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By Western blot analysis, we were able to show that the IE2-p86 protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1. Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling. By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant. This suggests a functional relevance of covalent modification by ubiquitin-homologous proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.
Phylogenetic Analyses Indicate an Atypical Nurse-to-Patient Transmission of Human Immunodeficiency Virus Type 1
Authors: Goujon Christophe P. , Schneider Véronique M. , Grofti Jaouad , Montigny Joëlle , Jeantils Vincent , Astagneau Pascal , Rozenbaum Willy , Lot Florence , Frocrain-Herchkovitch Claudie , Delphin Nathalie , Le Gal Frédéric , Nicolas Jean-Claude , Milinkovitch Michel C. , Dény Paul
Published: July 2002

A human immunodeficiency virus (HIV)-negative patient with no risk factor experienced HIV type 1 (HIV-1) primary infection 4 weeks after being hospitalized for surgery. Among the medical staff, only two night shift nurses were identified as HIV-1 seropositive. No exposure to blood was evidenced. To test the hypothesis of a possible nurse-to-patient transmission, phylogenetic analyses were conducted using two HIV-1 genomic regions (
pol
reverse transcriptase [RT] and
env
C2C4), each compared with reference strains and large local control sets (57 RT and 41 C2C4 local controls). Extensive analyses using multiple methodologies allowed us to test the robustness of phylogeny inference and to assess transmission hypotheses. Results allow us to unambiguously exclude one HIV-positive nurse and strongly suggest the other HIV-positive nurse as the source of infection of the patient.
Repression of the Integrated Papillomavirus E6/E7 Promoter Is Required for Growth Suppression of Cervical Cancer Cells
Authors: Francis Delicia A. , Schmid Susanne I. , Howley Peter M.
Published: July 2002

The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of “high-risk” HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA.
Tissue Sites of Persistent Infection and Active Replication of Equine Infectious Anemia Virus during Acute Disease and Asymptomatic Infection in Experimentally Infected Equids
Authors: Harrold Sharon M. , Cook Sheila J. , Cook R. Frank , Rushlow Keith E. , Issel Charles J. , Montelaro Ronald C.
Published: July 2002

Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAVWyoand the virulent cell-adapted strain designated EIAVPV. Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of the cellular viral burden. These quantitative studies demonstrate that tissues, rather than PBMC, constitute the predominant sites of virus replication during acute disease in infected equids and serve as resilient reservoirs of virus infection, even in the presence of highly effective immune responses that maintain a stringent control of virus replication in long-term inapparent carriers. Thus, these observations with EIAV, a predominantly macrophage-tropic lentivirus, highlight the role of tissues in sequestering lentiviral infections from host immune surveillance.
The T-Cell Receptor ζ Chain Contains Two Homologous Domains with Which Simian Immunodeficiency Virus Nef Interacts and Mediates Down-Modulation
Authors: Schaefer Todd M , Bell Ian , Fallert Beth A. , Reinhart Todd A.
Published: July 2002

We have recently demonstrated that simian immunodeficiency virus (SIV) Nef binds to the ζ chain of the T-cell receptor (TCR), leading to its down-modulation from T-cell surfaces (I. Bell, C. Ashman, J. Maughan, E. Hooker, F. Cook, and T. A. Reinhart, J. Gen. Virol. 79:2717–2727, 1998). Using a panel of human as well as rhesus macaque TCR ζ cytoplasmic domain mutants, we have identified in this report two linear peptides in the cytoplasmic domain of TCR ζ which independently interact with SIV Nef. Each SIV Nef interaction domain was sufficient in the absence of the other for interaction with SIV Nef in a yeast two-hybrid assay. In parallel, we demonstrated that Nef down-modulation of CD8-TCR ζ fusion proteins containing full-length or truncated portions of the TCR ζ cytoplasmic domain occurs in transiently transfected 293T cells. Furthermore, using proteins expressed inEscherichia coli, a glutathioneS-transferase–Nef fusion protein coprecipitated histidine-tagged portions of the TCR ζ cytoplasmic domain which contained SNID-1 or SNID-2. The peptides targeted by SIV Nef, YNELNL and YSEIGMKGERRR, are portions of the first and second of three immunoreceptor tyrosine-based activation motifs which are important in signal transduction, thymocyte development, and TCR biogenesis. These results demonstrate that SIV Nef binds to two distinct domains on TCR ζ in the absence of other T-cell-specific factors, and that interaction with either domain is sufficient to cause down-modulation of TCR ζ.
Induction of a Novel Cellular Homolog of Interleukin-10, AK155, by Transformation of T Lymphocytes with Herpesvirus Saimiri
Authors: Knappe Andrea , Hör Simon , Wittmann Sabine , Fickenscher Helmut
Published: July 2002

Although herpesvirus saimiri-transformed T lymphocytes retain multiple normal T-cell functions, only a few changes have been described. By subtractive hybridization, we have isolated a novel cellular gene,ak155, a sequence homolog of the interleukin-10 gene. Specifically herpesvirus saimiri-transformed T cells overexpressak155and secrete the protein into the supernatant. In other T-cell lines and in native peripheral blood cells, but not in B cells,ak155is transcribed at low levels. AK155 forms homodimers similarly to interleukin-10. As a lymphokine, AK155 may contribute to the transformed phenotype of human T cells after infection by herpesvirus saimiri.
Role of NF-κB and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein
Authors: Su Fei , Theodosis Christian N. , Schneider Robert J.
Published: July 2002

Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-α) and etopocide. Paradoxically, HBx activates members of the NF-κB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-α. We therefore examined the roles of NF-κB, c-Myc, and TNF-α in apoptotic killing of cells by HBx. RelA/NF-κB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-κB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-κB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-κB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-α. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis.
Modulation of Cellular and Viral Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Renne Rolf , Barry Chris , Dittmer Dirk , Compitello Nicole , Brown Patrick O. , Ganem Don
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)—and from NF-κB-dependent reporter genes—was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.
Immune Response Induced by Airway Sensitization after Influenza A Virus Infection Depends on Timing of Antigen Exposure in Mice
Authors: Yamamoto Naomi , Suzuki Shunsuke , Suzuki Yuzo , Shirai Akira , Nakazawa Masatoshi , Suzuki Motoyoshi , Takamasu Tetsuya , Nagashima Yoji , Minami Mutsuhiko , Ishigatsubo Yoshiaki
Published: July 2002

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.
DC-SIGN Interactions with Human Immunodeficiency Virus Type 1 and 2 and Simian Immunodeficiency Virus
Authors: Pöhlmann Stefan , Baribaud Frédéric , Lee Benhur , Leslie George J. , Sanchez Melissa D. , Hiebenthal-Millow Kirsten , Münch Jan , Kirchhoff Frank , Doms Robert W.
Published: July 2002

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.
Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection
Authors: Lagaye S. , Derrien M. , Menu E. , Coı̈to C. , Tresoldi E. , Mauclère P. , Scarlatti G. , Chaouat G. , Barré-Sinoussi F. , Bomsel M. ,   the European Network for the Study of In Utero Transmission of HIV-1
Published: July 2002

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.
Influenza A Virus NEP (NS2 Protein) Downregulates RNA Synthesis of Model Template RNAs
Authors: Bullido Rosario , Gómez-Puertas Paulino , Saiz Marı́a José , Portela Agustı́n
Published: July 2002

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.
Echoviruses Bind Heparan Sulfate at the Cell Surface
Authors: Goodfellow Ian G. , Sioofy Amir Babak , Powell Robert M. , Evans David J.
Published: July 2002

Some echoviruses (EV) that bind decay-accelerating factor (DAF) also bind cells of human and murine origins in a DAF-independent manner. Pretreatment of cells with heparinase 1 or heparin blocks the binding of radiolabeled virus to the cell surface, and heparin prevents infection of rhabdomyosarcoma cells by certain EV, including several low-passage clinical isolates of EV 6 and some EV that do not bind DAF. These studies suggest that heparan sulfate may be of in vivo relevance as an attachment molecule for EV.
Formation of Wild-Type and Chimeric Influenza Virus-Like Particles following Simultaneous Expression of Only Four Structural Proteins
Authors: Latham Theresa , Galarza Jose M.
Published: July 2002

We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of the VLPs. Immunoprecipitation of M1 protein from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines.
Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
Authors: Gonzalez Sonia L. , Stremlau Matt , He Xi , Basile John R. , Münger Karl
Published: July 2002

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB.
Duck Hepatitis B Virus Replication in Primary Bile Duct Epithelial Cells
Authors: Lee Jia-Yee , Culvenor Janetta G. , Angus Peter , Smallwood Richard , Nicoll Amanda , Locarnini Stephen
Published: July 2002

Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA , double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.