bluuebunny/crossref_metadata_2025 huggingface dataset:
Coronaviruses Maintain Viability despite Dramatic Rearrangements of the Strictly Conserved Genome Organization
Authors: de Haan Cornelis A. M. , Volders Haukeline , Koetzner Cheri A. , Masters Paul S. , Rottier Peter J. M.
Published: November 2002

Despite their high frequency of RNA recombination, the plus-strand coronaviruses have a characteristic, strictly conserved genome organization with the essential genes occurring in the order 5′-polymerase (
pol
)-S-E-M-N-3′. We have investigated the significance of this remarkable conservation by rearrangement of the murine coronavirus genome through targeted recombination. Thus, viruses were prepared with the following gene order: 5′-
pol
-S-M-E-N-3′, 5′-
pol
-S-N-E-M-3′, 5′-
pol
-M-S-E-N-3′, and 5′-
pol
-E-M-S-N-3′. All of these viruses were surprisingly viable, and most viruses replicated in cell culture with growth characteristics similar to those of the parental virus. The recombinant virus with the gene order 5′-
pol
-E-M-S-N-3′ was also tested for the ability to replicate in the natural host, the mouse. The results indicate that the canonical coronavirus genome organization is not essential for replication in vitro and in vivo. Deliberate rearrangement of the viral genes may be useful in the generation of attenuated coronaviruses, which due to their reduced risk of generating viable viruses by recombination with circulating field viruses, would make safer vaccines.
The Epstein-Barr Virus Immediate-Early Protein BZLF1 Regulates p53 Function through Multiple Mechanisms
Authors: Mauser Amy , Saito Shin'ichi , Appella Ettore , Anderson Carl W. , Seaman William T. , Kenney Shannon
Published: November 2002

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 is a transcriptional activator that mediates the switch between the latent and the lytic forms of EBV infection. It was previously reported that BZLF1 inhibits p53 transcriptional function in reporter gene assays. Here we further examined the effects of BZLF1 on p53 function by using a BZLF1-expressing adenovirus vector (AdBZLF1). Infection of cells with the AdBZLF1 vector increased the level of cellular p53 but prevented the induction of p53-dependent cellular target genes, such as p21 and MDM2. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. Thus, the inhibitory effects of BZLF1 on p53 transcriptional function cannot be explained by its effects on p53 phosphorylation, acetylation, or DNA binding activity. BZLF1 substantially reduced the level of cellular TATA binding protein (TBP) in both normal human fibroblasts and A549 cells, and the inhibitory effects of BZLF1 on p53 transcriptional function could be partially rescued by the overexpression of TBP. Thus, BZLF1 has numerous effects on p53 posttranslational modification but may inhibit p53 transcriptional function in part through an indirect mechanism involving the suppression of TBP expression.
Mechanism of De Novo Initiation by the Hepatitis C Virus RNA-Dependent RNA Polymerase: Role of Divalent Metals
Authors: Ranjith-Kumar C. T. , Kim Young-Chan , Gutshall Les , Silverman Carol , Khandekar Sanjay , Sarisky Robert T. , Kao C. Cheng
Published: November 2002

We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg
2+
and Mn
2+
gave rise to significant amounts of synthesis. Mg
2+
and Mn
2+
affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn
2+
significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn
2+
reduced the

K
m

for the initiation nucleotide, a GTP, from 103 to 3 μM. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn
2+
-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.
Requirements for De Novo Initiation of RNA Synthesis by Recombinant Flaviviral RNA-Dependent RNA Polymerases
Authors: Ranjith-Kumar C. T. , Gutshall Les , Kim Min-Ju , Sarisky Robert T. , Kao C. Cheng
Published: November 2002

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg
2+
and Mn
2+
on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn
2+
was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn
2+
. These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.
Healthy Skin of Many Animal Species Harbors Papillomaviruses Which Are Closely Related to Their Human Counterparts
Authors: Antonsson Annika , Hansson Bengt Göran
Published: November 2002

Papillomaviruses associated with clinical symptoms have been found in many vertebrate species. In this study, we have used an L1 gene consensus PCR test designed to detect a broad spectrum of human skin papillomaviruses to analyze swab samples from healthy skin of 111 animals belonging to 19 vertebrate species. In eight of the species, papillomavirus DNA was found with the following prevalences: chimpanzees, 9 of 11 samples positive; gorillas, 3 of 4; long-tailed macaques, 14 of 16; spider monkeys, 2 of 2; ruffed lemurs, 1 of 2; cows, 6 of 10; European elks, 4 of 4; aurochs, 1 of 1. In total, 53 new putative animal papillomavirus types were found. The results show that skin papillomaviruses can be detected in healthy skin from many different animal species and are sufficiently related genetically to their human counterparts to be identified by a human skin papillomavirus primer set (FAP59 and FAP64).
The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells
Authors: Mauser Amy , Holley-Guthrie Elizabeth , Zanation Adam , Yarborough Wendall , Kaufmann William , Klingelhutz Aloysius , Seaman William T. , Kenney Shannon
Published: November 2002

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G
1
/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G
1
/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.
Transcriptional Regulation of Porcine Endogenous Retroviruses Released from Porcine and Infected Human Cells by Heterotrimeric Protein Complex NF-Y and Impact of Immunosuppressive Drugs
Authors: Scheef Gregor , Fischer Nicole , Flory Egbert , Schmitt Isabel , Tönjes Ralf R.
Published: November 2002

Recent studies revealed a significant promoter activity of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) in different human and mammalian cell lines, which is mediated by a 39-bp repeat located in the U3 region in different numbers, representing an enhancer (G. Scheef, N. Fischer, U. Krach, and R. R. Tönjes, J. Virol. 75:6933-6940, 2001). A statistical transcription factor analysis revealed putative binding sites for the CCAAT-binding transcription factor NF-Y inside the 39-bp repeat. Specific binding of NF-Y to the repeat sequence was demonstrated by electrophoretic mobility shift assays and supershift assays with specific antibodies directed against the three subunits of NF-Y. To identify further transcription-regulating elements, genetically modified LTRs lacking the repeat box, U3, R, or U5 were investigated. The results indicated a strong inhibitory element in the R region, as the deletion of R caused a significantly increased promoter activity. Since PERV might play a potential role in the application of xenogeneic cell therapy and xenotransplantation techniques, we have investigated whether immunosuppressive drugs that are routinely used in transplantation medicine have an impact on the promoter activity. Neither cyclosporine nor prednisolone had any influence on the promoter strength of the PERV LTRs. By performing a real-time PCR we were able to compare the proviral loads of porcine and infected human cells as well as the amount of released virions, which revealed a direct link between LTR activity and the number of released retroviruses.
Differential Requirements for Activation of Integrated and Transiently Transfected Human T-Cell Leukemia Virus Type 1 Long Terminal Repeat
Authors: Okada Masahiko , Jeang Kuan-Teh
Published: November 2002

Adult T-cell leukemia (ATL) cells contain integrated human T-cell leukemia virus type 1 (HTLV-1) proviruses. Although the exact sequence of events leading to the development of ATL remains incompletely resolved, expression of the integrated HTLV-1 long terminal repeat (LTR) is likely required at some point during the process of T-cell transformation. While much has been learned about the regulated expression of transiently transfected LTR reporter plasmids, an analysis of factors required for expression of chromosomally integrated HTLV-1 LTR has not been done. Here, we have constructed CHOK1 and HeLa cells that contain an integrated HTLV-1 LTR-luciferase gene. Using these cells, we have compared the requirements for activation of transiently transfected versus stably integrated HTLV-1 LTR. We observed different requirements for CREB, p300, and P/CAF in the expression of transiently transfected versus stably integrated HTLV-1 LTR. Furthermore, with dominant-negative mutants of CREB, p300, and P/CAF, we found that activation of integrated HTLV-1 LTR by an ambient stress signal, UV-C, proceeds through a path mechanistically distinct from that used by viral oncoprotein, Tax. Our findings point to additional complexities in the regulated expression of HTLV-1 proviruses compared with those hitherto revealed through transfection studies.
Transcriptional Regulation of the K1 Gene Product of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Bowser Brian S. , DeWire Scott M. , Damania Blossom
Published: November 2002

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to be a transforming protein capable of inducing morphological changes and focus formation in rodent fibroblasts. K1 can activate B-cell receptor (BCR) signaling and upregulate activity of the NFAT and NF-κB transcription factors. In order to understand the regulation of K1 gene expression, we have analyzed sequences upstream of the K1 gene to identify the K1 promoter element. We have performed 5′ rapid amplification of cDNA ends as well as a nuclease protection assay to map the transcriptional start site of the KSHV K1 transcript. The K1 transcriptional start site lies 75 bp upstream of the translation start site. Sequences upstream of the K1 gene were characterized for their ability to activate a luciferase reporter gene in 293 epithelial cells, KSHV-negative B cells (BJAB), KSHV-positive B cells (BCBL-1), and KS tumor-derived endothelial cells (SLK-KS
−
). We found that a 125-bp sequence upstream of the K1 transcript start site was sufficient to fully activate the luciferase reporter gene in all cell types tested. In addition, the viral transcription factor KSHV Orf50/Rta was capable of further activating this promoter element in 293, BJAB, and BCBL-1 cells but not in SLK-KS
−
cells. Promoter constructs containing additional sequences upstream of the 125-bp element did not show further augmentation of transcription in the presence or absence of KSHV Orf50.
Broad Specificity of Virus-Specific CD4+T-Helper-Cell Responses in Resolved Hepatitis C Virus Infection
Authors: Day Cheryl L. , Lauer Georg M. , Robbins Gregory K. , McGovern Barbara , Wurcel Alysse G. , Gandhi Rajesh T. , Chung Raymond T. , Walker Bruce D.
Published: November 2002

Vigorous virus-specific CD4+T-helper-cell responses are associated with successful control of hepatitis C virus (HCV) and other human viral infections, but the breadth and specificity of responses associated with viral containment have not been defined. To address this we evaluated the HCV-specific CD4+T-helper-cell response in HCV antibody-positive persons who lack detectable plasma viremia, and compared this response to that in persons with chronic HCV infection. Peripheral blood mononuclear cells were stimulated with HCV proteins, followed by measurement of HCV-specific CD4+-T-cell responses to a comprehensive set of overlapping HCV peptides by intracellular gamma interferon production. In three persons with resolved HCV infection studied in detail, 13 to 14 epitopes were targeted, but none was recognized by all three. The 37 defined epitopes were predominantly distributed among the HCV proteins core, NS3, NS4, and NS5. In an expanded analysis of responses to these proteins in persons with resolved infection, an average of 10 epitopes was targeted, whereas in persons with chronic viremia never was more than one epitope targeted (P< 0.001). This comprehensive analysis of the breadth and specificity of HCV-specific T-helper-cell responses indicates that up to 14 viral epitopes can be simultaneously targeted by circulating virus-specific CD4+T helper cells in a controlled human viral infection. Moreover, these data provide important parameters for evaluation of candidate HCV vaccines, and provide rationale for immunotherapy in chronic HCV infection.
Trachea, Lung, and Tracheobronchial Lymph Nodes Are the Major Sites Where Antigen-Presenting Cells Are Detected after Nasal Vaccination of Mice with Human Papillomavirus Type 16 Virus-Like Particles
Authors: Balmelli Carole , Demotz Stéphane , Acha-Orbea Hans , De Grandi Pierre , Nardelli-Haefliger Denise
Published: November 2002

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4+T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.
Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD8+-T-Cell Responses for Groups of HIV-1-Infected Individuals with Different HLA-B*35 Genotypes
Authors: Jin Xia , Gao Xiaojiang , Ramanathan, Murugappan , Deschenes Geoffrey R. , Nelson George W. , O'Brien Stephen J. , Goedert James J. , Ho David D. , O'Brien Thomas R. , Carrington Mary
Published: November 2002

Human immunodeficiency virus type 1 (HIV-1)-infected individuals with HLA-B*35 allelic variants B*3502/3503/3504/5301 (B*35-Px) progress more rapidly to AIDS than do those with B*3501 (B*35-PY). The mechanisms responsible for this phenomenon are not clear. To examine whether cellular immune responses may differ according to HLA-B*35 genotype, we quantified HIV-1-specific CD8+-T-cell (CTL) responses using an intracellular cytokine-staining assay with specimens from 32 HIV-1-positive individuals who have B*35 alleles. Among them, 75% had CTL responses to Pol, 69% had CTL responses to Gag, 50% had CTL responses to Nef, and 41% had CTL responses to Env. The overall magnitude of CTL responses did not differ between patients bearing B*35-Px genotypes and those bearing B*35-PY genotypes. A higher percentage of Gag-specific CTL was associated with lower HIV-1 RNA levels (P= 0.009) in individuals with B*35-PY. A negative association between CTL activity for each of the four HIV antigens and viral load was observed among individuals with B*35-PY, and the association reached significance for Gag. No significant relationship between CTL activity and viral load was observed in the B*35-Px group. The relationship between total CTL activity and HIV RNA among B*35-Px carriers differed significantly from that among B*35-PY carriers (P< 0.05). The data are consistent with the hypothesis that higher levels of virus-specific CTL contribute to protection against HIV disease progression in infected individuals with B*35-PY, but not in those with B*35-Px.
Selectively ReducedtatmRNA Heralds the Decline in Productive Human Immunodeficiency Virus Type 1 Infection in Monocyte-Derived Macrophages
Authors: Sonza Secondo , Mutimer Helen P. , O'Brien Kate , Ellery Philip , Howard Jane L. , Axelrod Jonathan H. , Deacon Nicholas J. , Crowe Suzanne M. , Purcell Damian F. J.
Published: November 2002

The transcription and splicing of human immunodeficiency virus type 1 (HIV-1) mRNA in primary blood monocyte-derived macrophages (MDM) and CD4+peripheral blood lymphocytes (PBL) were compared to determine whether any differences might account for the slower noncytopathic infection of cells of the macrophage lineage. The expression of regulatory mRNAs during acute infection of MDM was delayed by about 12 h compared to that of PBL. In each cell type, an increase in spliced viral mRNAs slightly preceded virus production from the culture. Following the peak of productive infection, there was a proportional decrease in the expression of all regulatory mRNAs detected in PBL. In MDM, a dramatic additional decrease specifically in thetatmRNA species heralded a reduction in virus production. This decline intatmRNA was reflected by a concomitant decrease in Tat activity in the cells and occurred with the same kinetics irrespective of the age of the cells when infected. Addition of exogenous Tat protein elicited a burst of virus production from persistently infected MDM, suggesting that the decrease in virus production from the cultures is a consequence of the reduction intatmRNA levels. Our results show that modulation of HIV-1 mRNAs in macrophages during long-term infection, which is dependent on the period of infection rather than cell differentiation or maturation, results in a selective reduction of Tat protein levels at the commencement of a persistent, less productive phase of infection. Determination of the mechanism of this mRNA modulation may lead to novel targets for control of replication in these important viral reservoirs.
Newcastle Disease Virus HN Protein Alters the Conformation of the F Protein at Cell Surfaces
Authors: McGinnes Lori W. , Gravel Kathryn , Morrison Trudy G.
Published: November 2002

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F
0
and F
1
forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.
Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction
Authors: Ros Carlos , Burckhardt Christoph J. , Kempf Christoph
Published: November 2002

The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycinA1and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitorsN-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitorNα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.
Identification of Immunogenic Hot Spots within Plum Pox Potyvirus Capsid Protein for Efficient Antigen Presentation
Authors: Fernández-Fernández M. Rosario , Martínez-Torrecuadrada Jorge L. , Roncal Fernando , Domínguez Elvira , García Juan Antonio
Published: November 2002

PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-γ, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.
RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts
Authors: Olsthoorn R. C. L. , Bruyere A. , Dzianott A. , Bujarski J. J.
Published: November 2002

A model system of a single-stranded trisegment
Brome mosaic bromovirus
(BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3′ noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.
In Vitro Activity of the Baculovirus Late Expression Factor LEF-5
Authors: Guarino Linda A. , Dong Wen , Jin Jianping
Published: November 2002

The baculovirus late expression factor LEF-5 has a zinc ribbon that is homologous to a domain in the eukaryotic transcription elongation factor SII. To determine whether LEF-5 is an elongation factor, we purified it from a bacterial overexpression system and added it to purified baculovirus RNA polymerase. LEF-5 increased transcription from both late and very late viral promoters. Two acidic residues within the zinc ribbon were essential for stimulation. Unlike SII, however, LEF-5 did not appear to enable RNA polymerase to escape from intrinsic pause sites. Furthermore, LEF-5 did not increase transcription in the presence of small DNA-binding ligands that inhibit elongation in other systems or viral DNA-binding proteins which inhibit the baculovirus RNA polymerase. Exonuclease activity assays revealed that baculovirus RNA polymerase has an intrinsic exonuclease activity, but this was not increased by the addition of LEF-5. Initiation assays and elongation assays using heparin to prevent reinitiation indicated that LEF-5 was active only in the absence of heparin. Taken together, these results suggest that LEF-5 functions as an initiation factor and not as an elongation factor.
Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex
Authors: Kubota Toru , Yokosawa Noriko , Yokota Shin-ichi , Fujii Nobuhiro
Published: November 2002

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione
S
-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.
C-Terminal Region of STAT-1α Is Not Necessary for Its Ubiquitination and Degradation Caused by Mumps Virus V Protein
Authors: Yokosawa Noriko , Yokota Shin-ichi , Kubota Toru , Fujii Nobuhiro
Published: November 2002

Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-α) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-α-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.
Effects of Membrane Potential and Sphingolipid Structures on Fusion of Semliki Forest Virus
Authors: Samsonov Andrey V. , Chatterjee Prodyot K. , Razinkov Vladimir I. , Eng Christina H. , Kielian Margaret , Cohen Fredric S.
Published: November 2002

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the
trans
side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C
2
-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C
2
-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.
Detection of the Potyviral Genome-Linked Protein VPg in Virions and Its Phosphorylation by Host Kinases
Authors: Puustinen Pietri , Rajamäki Minna-Liisa , Ivanov Konstantin I. , Valkonen Jari P. T. , Mäkinen Kristiina
Published: November 2002

The multifunctional genome-linked protein (VPg) of
Potato virus A
(PVA; genus
Potyvirus
) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species
Solanum commersonii
and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of
S. commersonii
differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.
Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein
Authors: Chatterjee Prodyot K. , Eng Christina H. , Kielian Margaret
Published: November 2002

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants,
srf-4
and
srf-5
, selected for growth in cholesterol-depleted cells. Like the previously isolated
srf-3
mutant (E1 proline 226 to serine), the phenotypes of the
srf-4
and
srf-5
mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like
srf-3
,
srf-4
and
srf-5
show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike
srf-3
,
srf-4
and
srf-5
showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both
srf-4
and
srf-5
formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the
srf-3
mutant. All three
srf
mutations lie in the same domain of E1, but the
srf-4
and
srf-5
mutations are spatially separated from
srf-3
. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the
srf-4
and
srf-5
mutations identify novel regions of E1 that are distinct from the fusion peptide and
srf-3
region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.
Similar Regulation of Cell Surface Human T-Cell Leukemia Virus Type 1 (HTLV-1) Surface Binding Proteins in Cells Highly and Poorly Transduced by HTLV-1-Pseudotyped Virions
Authors: Jones Kathryn S. , Nath Manisha , Petrow-Sadowski Cari , Baines Andrea C. , Dambach Megan , Huang Ying , Ruscetti Francis W.
Published: November 2002

Little is known about the requirements for human T-cell leukemia virus type 1 (HTLV-1) entry, including the identity of the cellular receptor(s). Previous studies have shown that although the HTLV receptor(s) are widely expressed on cell lines of various cell types from different species, cell lines differ dramatically in their susceptibility to HTLV-Env-mediated fusion. Human cells (293, HeLa, and primary CD4+T cells) showed higher levels of binding at saturation than rodent (NIH 3T3 and NRK) cells to an HTLV-1 SU immunoadhesin. A direct comparison of the binding of the HTLV-1 surface glycoprotein (SU) immunoadhesin and transduction by HTLV-1 pseudotyped virus revealed parallels between the level of binding and the titer for various cell lines. When cells were treated with phorbol myristate acetate (PMA), which down-modulates a number of cell surface molecules, the level of SU binding was markedly reduced. However, PMA treatment only slightly reduced the titer of murine leukemia virus(HTLV-1) on both highly susceptible and poorly susceptible cells. Treatment of target cells with trypsin greatly reduced binding, indicating that the majority of HTLV SU binding is to proteins. Polycations, which enhance the infectivity of several other retroviruses, inhibited HTLV-1 Env-mediated binding and entry on both human and rodent cells. These results suggest that factors other than the number of primary binding receptors are responsible for the differences in the titers of HTLV-1 pseudotypes between highly susceptible cells and poorly susceptible cells.
Comparative Vaccine Studies in HLA-A2.1-Transgenic Mice Reveal a Clustered Organization of Epitopes Presented in Hepatitis C Virus Natural Infection
Authors: Himoudi Nourredine , Abraham Jean-Daniel , Fournillier Anne , Lone Yu Chun , Joubert Aurélie , De Beeck Anne Op , Freida Delphine , Lemonnier François , Kieny Marie Paule , Inchauspé Geneviève
Published: November 2002

A polyepitopic CD8
+
-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.
Tomato Bushy Stunt Virus
Genomic RNA Accumulation Is Regulated by Interdependent
cis
-Acting Elements within the Movement Protein Open Reading Frames
Authors: Park Jong-Won , Desvoyes Bénédicte , Scholthof Herman B.
Published: November 2002

This study on
Tomato bushy stunt virus
identified and defined three previously unknown regulatory sequences involved in RNA accumulation that are located within the 3′-proximal nested movement protein genes
p22
and
p19
. The first is a 16-nucleotide (nt) element termed III-A that is positioned at the very 3′ end of
p22
and is essential for RNA accumulation. Approximately 300 nt upstream of III-A resides an ∼80-nt inhibitory element (IE) that is obstructive to replication only in the absence of a third regulatory element of ∼30 nt (SUR-III) that is positioned immediately upstream of III-A. Inspection of the nucleotide sequences predicted that III-A and SUR-III can form looped hairpins. A comparison of different tombusviruses showed, in each case, conservation for potential base pairing between the two predicted hairpin-loops. Insertion of a spacer adjacent to the predicted hairpins had no or a minimal effect on RNA accumulation, whereas an insertion in the putative III-A loop abolished genomic RNA multiplication. We conclude that the sequences composing the predicted III-A and SUR-III hairpin-loops are crucial for optimal RNA accumulation and that the inhibitory effect of IE surfaces when the alleged interaction between SUR-III and III-A is disturbed.
General and Specific Alterations in Programming of Global Viral Gene Expression during Infection by VP16 Activation-Deficient Mutants of Herpes Simplex Virus Type 1
Authors: Yang William C. , Devi-Rao G. V. , Ghazal Peter , Wagner Edward K. , Triezenberg Steven J.
Published: November 2002

During productive infection by herpes simplex virus 1 (HSV-1), viral gene expression occurs in a temporally regulated cascade in which transcription of the viral immediate-early (IE) genes is strongly stimulated by the virion protein VP16. We have employed an oligonucleotide microarray to examine the effect of VP16 mutations on the overall pattern of viral gene expression following infection of HeLa cells. This microarray detects essentially all HSV-1 transcripts with relative and absolute levels correlating well with known kinetics of expression. This analysis revealed that deletion of the VP16 activation domain sharply reduced overall viral gene expression; moreover, the pattern of this reduced expression varied greatly from the pattern of a wild-type (wt) infection. However, when this mutant virus was delivered at a high multiplicity of infection or in the presence of the cellular stress inducer hexamethylene bisacetamide, expression was largely restored to the wt levels and pattern. Infection with virions that deliver wt VP16 protein at the start of infection but synthesize only truncated VP16 resulted in a normal kinetic cascade. This suggests that newly synthesized VP16 does not play a significant role in the expression of later classes of transcripts. The VP16 activation domain comprises two subregions. Deletion of the C-terminal subregion resulted in minimal changes in the level and profile of gene expression compared to a normal (wt) cascade. In contrast, deletion of the N-terminal subregion reduced the overall expression levels and skewed the relative levels of IE transcripts but did not significantly alter the kinetic pattern of early and late transcript expression. We conclude that the general activation of IE gene transcription by VP16, but not the specific ratios of IE transcripts, is necessary for the subsequent ordered expression of viral genes. Moreover, this report establishes the feasibility of microarray analysis for globally assessing viral gene expression programs as a function of the conditions of infection.
Construction and Characterization of Adenovirus Serotype 5 Packaged by Serotype 3 Hexon
Authors: Wu Hongju , Dmitriev Igor , Kashentseva Elena , Seki Toshiro , Wang Minghui , Curiel David T.
Published: November 2002

Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.