bluuebunny/crossref_metadata_2025 huggingface dataset:
A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes
Authors: Kämper Nadine , Day Patricia M. , Nowak Thorsten , Selinka Hans-Christoph , Florin Luise , Bolscher Jan , Hilbig Lydia , Schiller John T. , Sapp Martin
Published: December 2005

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides.
UL54
-Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture
Authors: Schwartz Jennifer A. , Brittle Elizabeth E. , Reynolds Ashley E. , Enquist Lynn W. , Silverstein Saul J.
Published: December 2005

The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed
UL54
null mutations in a PRV bacterial artificial chromosome using sugar suicide and λRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in
trans
by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of
UL54
in the virus vJSΔ54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSΔ54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSΔ54. UL54 appears to regulate
UL53
and
UL52
at the transcriptional level as their respective RNAs are decreased in cells infected with vJSΔ54. Interestingly, vJSΔ54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSΔ54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.
7a Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cellular Protein Synthesis and Activates p38 Mitogen-Activated Protein Kinase
Authors: Kopecky-Bromberg Sarah A. , Martinez-Sobrido Luis , Palese Peter
Published: December 2005

It was recently shown that the 7a protein of severe acute respiratory syndrome coronavirus induces biochemical changes associated with apoptosis. In this study, the mechanism by which the 7a protein induces apoptosis was examined. The 7a protein was tested for the ability to inhibit cellular gene expression because several proapoptotic viral proteins with this function have previously been identified. 7a protein inhibited expression of luciferase from an mRNA construct that specifically measures translation, whereas inhibitors of transcription and nucleocytoplasmic transport did not. The inhibition of translation and other cellular processes of gene expression have been associated with the induction of a stress response in cells. Western blot analysis using phosphospecific antibodies indicated that 7a protein activated p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal protein kinase/stress-activated protein kinase. Taken together, these data indicate that the induction of apoptosis by the 7a protein may be related to its ability to inhibit cellular translation and activate p38 MAPK.
Involvement of Template-Activating Factor I/SET in Transcription of Adenovirus Early Genes as a Positive-Acting Factor
Authors: Haruki Hirohito , Okuwaki Mitsuru , Miyagishi Makoto , Taira Kazunari , Nagata Kyosuke
Published: December 2005

The adenovirus genome complexed with viral core protein VII (adenovirus DNA-protein VII complex) at least is the bona fide template for transcription of adenovirus early genes. It is believed that the highly basic protein VII, like cellular histones, is a negative regulator for genome functions. Analyses with in vitro replication and transcription systems using the adenovirus DNA-protein VII complex have revealed that remodeling of the complex is crucial for efficient DNA replication and transcription. We identified host acidic proteins, template-activating factor I (TAF-I), TAF-II, and TAF-III as stimulatory factors for replication from the adenovirus DNA-protein VII complex. Recently, it was reported that the adenovirus DNA interacts with TAF-I and pp32, another host acidic protein (Y. Xue, J. S. Johnson, D. A. Ornelles, J. Lieberman, and D. A. Engel, J. Virol. 79:2474-2483, 2005). We found that TAF-I interacts and colocalizes with protein VII in adenovirus-infected cells during the early phases of infection, but pp32 does not. Although pp32 had the potential ability to interact with protein VII, pp32 did not remodel the adenovirus DNA-protein VII complex in vitro. Small interfering RNA-mediated knockdown of TAF-I expression leads to the delay of the transcription timing of early genes. These results provide evidence that TAF-I plays an important role in the early stages of the adenovirus infection cycle.
Naïve and Memory Cell Turnover as Drivers of CCR5-to-CXCR4 Tropism Switch in Human Immunodeficiency Virus Type 1: Implications for Therapy
Authors: Ribeiro Ruy M. , Hazenberg Mette D. , Perelson Alan S. , Davenport Miles P.
Published: December 2005

Early human immunodeficiency virus infection is characterized by the predominance of CCR5-tropic (R5) virus. However, in many individuals CXCR4-tropic (X4) virus appears in late infection. The reasons for this phenotypic switch are unclear. The patterns of chemokine receptor expression suggest that X4 and R5 viruses have a preferential tropism for naïve and memory T cells, respectively. Since memory cells divide approximately 10 times as often as naïve cells in uninfected individuals, a tropism for memory cells in early infection may provide an advantage. However, with disease progression both naïve and memory cell division frequencies increase, and at low CD4 counts, the naïve cell division frequency approaches that of memory cells. This may provide a basis for the phenotypic switch from R5 to X4 virus observed in late infection. We show that a model of infection using observed values for cell turnover supports this mechanism. The phenotypic switch from R5 to X4 virus occurs at low CD4 counts and is accompanied by a rapid rise in viral load and drop in CD4 count. Thus, low CD4 counts are both a cause and an effect of X4 virus dominance. We also investigate the effects of different antiviral strategies. Surprisingly, these results suggest that both conventional antiretroviral regimens and CCR5 receptor-blocking drugs will promote R5 virus over X4 virus.
Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging
Authors: Bleker Svenja , Pawlita Michael , Kleinschmidt Jürgen A.
Published: December 2005

Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol.
74:
8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.
Mutations on the External Surfaces of Adeno-AssociatedVirus Type 2 Capsids That Affect Transduction andNeutralization
Authors: Lochrie Michael A. , Tatsuno Gwen P. , Christie Brian , McDonnell Jennifer Wellman , Zhou Shangzhen , Surosky Richard , Pierce Glenn F. , Colosi Peter
Published: December 2005

Mutations
were made at 64 positions on the external surface of the
adeno-associated virus type 2 (AAV-2) capsid in regions expected to
bind antibodies. The 127 mutations included 57 single alanine
substitutions, 41 single nonalanine substitutions, 27 multiple
mutations, and 2 insertions. Mutants were assayed for capsid synthesis,
heparin binding, in vitro transduction, and binding and neutralization
by murine monoclonal and human polyclonal antibodies. All mutants made
capsid proteins within a level about 20-fold of that made by the wild
type. All but seven mutants bound heparin as well as the wild type.
Forty-two mutants transduced human cells at least as well as the wild
type, and 10 mutants increased transducing activity up to ninefold more
than the wild type. Eighteen adjacent alanine substitutions diminished
transduction from 10- to 100,000-fold but had no effect on heparin
binding and define an area (dead zone) required for transduction that
is distinct from the previously characterized heparin receptor binding
site. Mutations that reduced binding and neutralization by a murine
monoclonal antibody (A20) were localized, while mutations that reduced
neutralization by individual human sera or by pooled human, intravenous
immunoglobulin G (IVIG) were dispersed over a larger area. Mutations
that reduced binding by A20 also reduced neutralization. However, a
mutation that reduced the binding of IVIG by 90% did not reduce
neutralization, and mutations that reduced neutralization by IVIG did
not reduce its binding. Combinations of mutations did not significantly
increase transduction or resistance to neutralization by IVIG. These
mutations define areas on the surface of the AAV-2 capsid that are
important determinants of transduction and antibody
neutralization.
Neutralization Escape Variants of Human Immunodeficiency Virus Type 1 Are Transmitted from Mother to Infant
Authors: Wu Xueling , Parast Adam B. , Richardson Barbra A. , Nduati Ruth , John-Stewart Grace , Mbori-Ngacha Dorothy , Rainwater Stephanie M. J. , Overbaugh Julie
Published: December 2005

Maternal passive immunity typically plays a critical role in protecting infants from new infections; however, the specific contribution of neutralizing antibodies in limiting mother-to-child transmission of human immunodeficiency virus type 1 is unclear. By examining cloned envelope variants from 12 transmission pairs, we found that vertically transmitted variants were more resistant to neutralization by maternal plasma than were maternal viral variants near the time of transmission. The vertically transmitted envelope variants were poorly neutralized by monoclonal antibodies biz, 2G12, 2F5, and 4E10 individually or in combination. Despite the fact that the infant viruses were among the most neutralization resistant in the mother, they had relatively few glycosylation sites. Moreover, the transmitted variants elicited de novo neutralizing antibodies in the infants, indicating that they were not inherently difficult to neutralize. The neutralization resistance of vertically transmitted viruses is in contrast to the relative neutralization sensitivity of viruses sexually transmitted within discordant couples, suggesting that the antigenic properties of viruses that are favored for transmission may differ depending upon mode of transmission.
Epstein-Barr Virus (EBV) Genome and Expression in Breast Cancer Tissue: Effect of EBV Infection of Breast Cancer Cells on Resistance to Paclitaxel (Taxol)
Authors: Arbach Hratch , Viglasky Viktor , Lefeu Florence , Guinebretière Jean-Marc , Ramirez Vanessa , Bride Nadège , Boualaga Nadia , Bauchet Thomas , Peyrat Jean-Philippe , Mathieu Marie-Christine , Mourah Samia , Podgorniak Marie-Pierre , Seignerin Jean-Marie , Takada Kenzo , Joab Irène
Published: December 2005

The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.
R5 Variants of Human Immunodeficiency Virus Type 1 Preferentially Infect CD62L−CD4+T Cells and Are Potentially Resistant to Nucleoside Reverse Transcriptase Inhibitors
Authors: Gondois-Rey Françoise , Biancotto Angelique , Fernandez Marcelo Antonio , Bettendroffer Lise , Blazkova Jana , Trejbalova Katerina , Pion Marjorie , Hirsch Ivan
Published: December 2005

The persistence of human immunodeficiency virus type 1 (HIV-1) in memory CD4+T cells is a major obstacle to the eradication of the virus with current antiretroviral therapy. Here, we investigated the effect of the activation status of CD4+T cells on the predominance of R5 and X4 HIV-1 variants in different subsets of CD4+T cells in ex vivo-infected human lymphoid tissues and peripheral blood mononuclear cells (PBMCs). In these cell systems, we examined the sensitivity of HIV replication to reverse transcriptase inhibitors. We demonstrate that R5 HIV-1 variants preferentially produced productive infection in HLA-DR−CD62L−CD4+T cells. These cells were mostly in the G1b phase of the cell cycle, divided slowly, and expressed high levels of CCR5. In contrast, X4 HIV-1 variants preferentially produced productive infection in activated HLA-DR+CD62L+CD4+T cells, which expressed high levels of CXCR4. The abilities of the nucleoside reverse transcriptase inhibitors (NRTI) zidovudine and lamivudine to stop HIV-1 replication were 20 times greater in activated T cells than in slowly dividing HLA-DR−CD62L−CD4+T cells. This result, demonstrated both in a highly physiologically relevant ex vivo lymphoid tissue model and in PBMCs, correlated with higher levels of thymidine kinase mRNA in activated than in slowly dividing HLA-DR−CD62L−CD4+T cells. The non-NRTI nevirapine was equally efficient in both cell subsets. The lymphoid tissue and PBMC-derived cell systems represent well-defined models which could be used as new tools for the study of the mechanism of resistance to HIV-1 inhibitors in HLA-DR−CD62L−CD4+T cells.
Hepatitis C Virus Induces Toll-Like Receptor 4 Expression, Leading to Enhanced Production of Beta Interferon and Interleukin-6
Authors: Machida Keigo , Cheng Kevin T. H. , Sung Vicky M.-H. , Levine Alexandra M. , Foung Steven , Lai Michael M. C.
Published: December 2005

Hepatitis C virus (HCV) induces inflammatory signals, leading to hepatitis, hepatocellular carcinomas, and lymphomas. The mechanism of HCV involvement in the host's innate immune responses has not been well characterized. In this study, we analyzed expression and regulation of the entire panel of toll-like receptors (TLRs) in human B cells following HCV infection in vitro. Among all of the TLRs (TLRs 1 to 10) examined, only TLR4 showed an altered expression (a three- to sevenfold up-regulation) after HCV infection. Peripheral blood mononuclear cells from HCV-infected individuals also showed a higher expression level of TLR4 compared with those of healthy individuals. HCV infection significantly increased beta interferon (IFN-β) and interleukin-6 (IL-6) secretion from B cells, particularly after lipopolysaccharide stimulation. The increased IFN-β and IL-6 production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HCV-induced cytokine production. Among all of the viral proteins, only NS5A caused TLR4 induction in hepatocytes and B cells. NS5A specifically activated the promoter of the TLR4 gene in both hepatocytes and B cells. In conclusion, HCV infection directly induces TLR4 expression and thereby activates B cells, which may contribute to the host's innate immune responses.
Uracil DNA Glycosylase Is Dispensable for Human Immunodeficiency Virus Type 1 Replication and Does Not Contribute to the Antiviral Effects of the Cytidine Deaminase Apobec3G
Authors: Kaiser Shari M. , Emerman Michael
Published: December 2005

It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human
UNG
−
/
−
cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G.
Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation
Authors: Ito Yoshinori , Kondo Eisei , Demachi-Okamura Ayako , Akatsuka Yoshiki , Tsujimura Kunio , Tanimoto Mitsune , Morishima Yasuo , Takahashi Toshitada , Kuzushima Kiyotaka
Published: December 2005

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.
Potent Neutralization of Hendra and Nipah Viruses by Human Monoclonal Antibodies
Authors: Zhu Zhongyu , Dimitrov Antony S. , Bossart Katharine N. , Crameri Gary , Bishop Kimberly A. , Choudhry Vidita , Mungall Bruce A. , Feng Yan-Ru , Choudhary Anil , Zhang Mei-Yun , Feng Yang , Wang Lin-Fa , Xiao Xiaodong , Eaton Bryan T. , Broder Christopher C. , Dimitrov Dimiter S.
Published: December 2005

Hendra virus
(HeV) and
Nipah virus
(NiV) are closely related emerging viruses comprising the
Henipavirus
genus of the
Paramyxovirinae
. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 μg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 μg/ml, and 98% neutralization required only 1.6 μg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.
The Amino-Terminal Domain of Bovine Viral Diarrhea Virus NproProtein Is Necessary for Alpha/Beta Interferon Antagonism
Authors: Gil Laura H. V. G. , Ansari Israrul H. , Vassilev Ventzislav , Liang Delin , Lai Vicky C. H. , Zhong Weidong , Hong Zhi , Dubovi Edward J. , Donis Ruben O.
Published: December 2005

The alpha/beta interferon (IFN-α/β) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-α/β secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-α/β response.Bovine viral diarrhea virus(BVDV) (genusPestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-α/β very inefficiently. Using a set of engineered cp BVDVs expressing mutant Nproand appropriate controls, we found that the IFN-α/β response to infection was dependent on Nproexpression and independent of viral replication efficiency. In order to investigate whether the protease activity of Nprois required for IFN-α/β antagonism, we engineered Npromutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22and H49substitutions abolished the ability of Nproto suppress IFN, whereas C69had no effect, suggesting that the structural integrity of the N terminus of Nprowas more important than its catalytic activity for IFN-α/β suppression. A catalytically active mutant with a change at a conserved Nproregion near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-α/β production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Nproin blocking IFN-α/β induction, but also implicate the amino-terminal domain of the protein in this function.
Immune Response to Individual Maedi-Visna VirusgagAntigens
Authors: Singh Inderpal , McConnell Ian , Blacklaws Barbara
Published: December 2005

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individualgagantigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all threegagantigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinantgagantigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+T lymphocytes. All threegagantigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.
Human Cytomegalovirus Immediate-Early 2 Protein IE86 Blocks Virus-Induced Chemokine Expression
Authors: Taylor R. Travis , Bresnahan Wade A.
Published: December 2005

The effect of human cytomegalovirus (HCMV) gene expression on cytokine (beta interferon) and chemokine (RANTES, MIG, MCP-2, MIP-1α, and interleukin-8) expression was examined. We demonstrate that HCMV gene expression is required to suppress the transcriptional induction of these cytokines and that the HCMV immediate-early 2 gene product IE86 can effectively block the expression of cytokines and proinflammatory chemokines during HCMV and Sendai virus infection. Additionally, we present data on viral mutants and ectopic protein expression which demonstrate that pp65, another identified HCMV cytokine antagonist, is not involved in regulating these proinflammatory cytokines. This is the first report to demonstrate that IE86 can act to suppress virus-induced proinflammatory cytokine transcript expression, extending the antiviral properties of this multifunctional viral protein.
Functional Analysis of the Triplex Proteins (VP19C and VP23) of Herpes Simplex Virus Type 1
Authors: Okoye Mercy E. , Sexton Gerry L. , Huang Eugene , McCaffery J. Michael , Desai Prashant
Published: December 2005

The triplex of herpesvirus capsids is a unique structural element. In herpes simplex virus type 1 (HSV-1), one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent VP5 molecules. The interaction between VP19C and VP23 was inferred by yeast cryoelectron microscopy studies and subsequently confirmed by the two-hybrid assay. In order to define the functional domains of VP19C and VP23, a Tn
7
-based transposon was used to randomly insert 15 bp into the coding regions of these two proteins. The mutants were initially screened for interaction in the yeast two-hybrid assay to identify the domains important for triplex formation. Using genetic complementation assays in HSV-1-infected cells, the domains of each protein required for virus replication were similarly uncovered. The same mutations that abolish interaction between these two proteins in the yeast two-hybrid assay similarly failed to complement the growth of the VP23- and VP19C-null mutant viruses in the genetic complementation assay. Some of these mutants were transferred into recombinant baculoviruses to analyze the effect of the mutations on herpesvirus capsid assembly in insect cells. The mutations that abolished the interaction in the yeast two-hybrid assay also abolished capsid assembly in insect cells. The outcome of these experiments showed that insertions in at least four regions and especially the amino terminus of VP23 abolished function, whereas the amino terminus of VP19C can tolerate transposon insertions. A novel finding of these studies was the ability to assemble herpesvirus capsids in insect cells using VP5 and VP19C that contained a histidine handle at their amino terminus.
Monoclonal Antibodies Targeting the HR2 Domain and the Region Immediately Upstream of the HR2 of the S Protein Neutralize In Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus
Authors: Lip Kuo-Ming , Shen Shuo , Yang Xiaoming , Keng Choong-Tat , Zhang Aihua , Oh Hsueh-Ling Janice , Li Zhi-Hong , Hwang Le-Ann , Chou Chih-Fong , Fielding Burtram C. , Tan Timothy H. P. , Mayrhofer Josef , Falkner Falko G. , Fu Jianlin , Lim Seng Gee , Hong Wanjin , Tan Yee-Joo
Published: December 2005

We have previously shown that an
Escherichia coli
-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289-3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.
Human Cytomegalovirus Protein pp71 Disrupts Major Histocompatibility Complex Class I Cell Surface Expression
Authors: Trgovcich Joanne , Cebulla Colleen , Zimmerman Pete , Sedmak Daniel D.
Published: December 2005

The human cytomegalovirus tegument protein pp71 is the product of the UL82 gene. Roles for pp71 in stimulating gene transcription, increasing infectivity of viral DNA, and the degradation of retinoblastoma family proteins have been described. Here we report a novel function for pp71 in limiting accumulation of cell surface major histocompatibility complex (MHC) class I complexes. MHC molecules were analyzed in glioblastoma cells exposed to a replication-defective adenovirus expressing UL82 (Adpp71) or after transient transfection of the UL82 gene. Accumulation of cell surface MHC class I levels diminished in a specific and dose-dependent manner after exposure to Adpp71 but not after exposure to an adenovirus expressing β-galactosidase (Adβgal). UL82 expression did not interfere with accumulation of either MHC class I heavy-chain transcript or protein, nor did UL82 expression correlate with markers of apoptosis. Rather, UL82 expression correlated with an increased proportion of MHC class I molecules exhibiting sensitivity to endoglycosidase H treatment. Finally, we show that, in cells infected with recombinant virus strain missing all of the unique short region MHC class I evasion genes, disruption of UL82 expression by short, interfering RNAs led to increased accumulation of cell surface MHC class I complexes. These findings support a novel role for HCMV pp71 in disruption of the MHC class I antigen presentation pathway.
Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts
Authors: Gonzalez Ramon , Huang Wenying , Finnen Renee , Bragg Courtney , Flint S. J.
Published: December 2005

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol.
76:
4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology
179:
806-814, 1990).
Natural History of Human Respiratory Syncytial Virus Inferred from Phylogenetic Analysis of the Attachment (G) Glycoprotein with a 60-Nucleotide Duplication
Authors: Trento Alfonsina , Viegas Mariana , Galiano Mónica , Videla Cristina , Carballal Guadalupe , Mistchenko Alicia S. , Melero José A.
Published: December 2005

A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen.
Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells
Authors: Guerra Susana , López-Fernández Luis A. , Pascual-Montano Alberto , Nájera José Luis , Zaballos Angel , Esteban Mariano
Published: December 2005

NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-κB1 and NF-κB2 as well as that of NF-κB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-κB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.
Consistent Patterns of Change during the Divergence of Human Immunodeficiency Virus Type 1 Envelope from That of the Inoculated Virus in Simian/Human Immunodeficiency Virus-Infected Macaques
Authors: Blay W.M. , Gnanakaran S. , Foley B. , Doria-Rose N. A. , Korber B. T. , Haigwood N. L.
Published: December 2005

We have analyzed changes to proviral Env gp120 sequences and the development of neutralizing antibodies (NAbs) during 1 year of simian/human immunodeficiency virus SHIV-89.6P infection in 11Macaca nemestrinamacaques. Seven macaques had significantenvdivergence from that of the inoculum, and macaques with greater divergence had higher titers of homologous NAbs. Substitutions in sequons encoding potential N-linked glycosylation sites (PNGs) were among the first to be established, although overall the total number of sequons did not increase significantly. The majority (19 of 23) of PNGs present in the inoculum were conserved in the sequences from all macaques. Statistically significant variations in PNGs occurred in multiple macaques within constrained regions we term “hot spots,” resulting in the selection of sequences more similar to the B consensus. These included additions on V1, the N-terminal side of V4, and the outer region of C2. Complex mutational patterns resulted in convergent PNG shifts in V2 and V5. Charge changes in Env V1V2, resulting in a net acidic charge, and a proline addition in V5 occurred in several macaques. Molecular modeling of the 89.6P sequence showed that the conserved glycans lie on the silent face of Env and that many are proximal to disulfide bonds, while PNG additions and shifts are proximal to the CD4 binding site. Nonsynonymous-to-synonymous substitution ratios suggest that these changes result from selective pressure. This longitudinal and cross-sectional study of mutations in human immunodeficiency virus (HIV)envin the SHIV background provides evidence that there are more constraints on the configuration of the glycan shield than were previously appreciated.
SYMMETRY AND SYMMETRY BREAKING FOR MINIMIZERS IN THE TRACE INEQUALITY
Authors: LAMI DOZO ENRIQUE J. , TORNÉ OLAF
Published: December 2005

We consider symmetry properties of minimizers in the variational characterization of the best constant in the trace inequality [Formula: see text] in the ball Bρ of radius ρ. When p is fixed, minimizers in this problem can be radial or non-radial depending on the parameters q and ρ. We prove that there is a global radial function u0 > 0, with u0 independent of q, such that any radial minimizer is a multiple of the restriction of u0 to Bρ. Next, we prove that if either q or ρ is sufficiently large, then the minimizers are non-radial. In the case when p = 2, we consider a generalization of the minimization problem and improve some of the above symmetry results. We also present some numerical results describing the exact values of q and ρ for which radial symmetry breaking occurs.
PARTIAL DIFFERENTIAL EQUATIONS OF ELLIPTIC TYPE IN NONSMOOTH DOMAINS
Authors: RODRIGUES HELDER CANDIDO
Published: December 2005

This paper studies the problem -Δu + λu = up in nonsmooth domains with mixed boundary conditions. Special attention will be given here to the critical case and to domains which have no further regularity than a Lipschitzian boundary. For such domains, we obtain a generalized version of Cherrier's inequality and prove an existence result. This was achieved by using an extended definition of the manifold.
DESCRIPTION OF THE REPRESENTATIONS OF THE ALGEBRAS GENERATED BY FOUR LINEARLY RELATED IDEMPOTENTS
Authors: STRELETS ALEXANDER
Published: December 2005

In this paper we find a description, up to equivalence, of all irreducible representations for a class of algebras generated by four linearly related idempotents.
Leptin and CCK selectively activate vagal afferent neurons innervating the stomach and duodenum
Authors: Peters J. H. , Ritter R. C. , Simasko S. M.
Published: December 2005

The hormone leptin and the gut peptide CCK synergistically interact to enhance the process of satiation. Although this interaction may occur at several levels of the neuroaxis, our previous results indicate that leptin can specifically enhance the satiation effect of CCK by acting on subdiaphragmatic vagal afferent neurons. Because of this localized action, we hypothesized that a high proportion of vagal afferent neurons innervating the stomach or duodenum would be responsive to leptin and/or CCK. To test this hypothesis, we measured changes in cytosolic calcium levels induced by leptin and CCK in cultured nodose ganglion neurons labeled with a retrograde neuronal tracer injected into either the stomach or the duodenum. In the neurons labeled from the stomach, CCK activated 74% (39 of 53) compared with only 35% (34 of 97) of nonlableled cells. Of the CCK-responsive neurons 60% (18 of 30) were capsaicin-sensitive. Leptin activated 42% (22 of 53) of the stomach innervating neurons compared with 26% of nonlabeled neurons. All of the leptin-sensitive neurons labeled from the stomach also responded to CCK. In the neurons labeled from the duodenum, CCK activated 71% (20 of 28). Of these CCK-responsive neurons 80% (12 of 15) were capsaicin sensitive. Leptin activated 46% (13 of 28) of these duodenal innervating neurons, of which 89% (8 of 9) were capsaicin-sensitive. Among neurons labeled from the duodenum 43% (12 of 28) were responsive to both leptin and CCK, compared with only 15% (15 of 97) of unlabeled neurons. Our results support the hypothesis that vagal afferent sensitivity to CCK and leptin is concentrated in neurons that innervate the stomach and duodenum. These specific visceral afferent populations are likely to comprise a substrate through which acute leptin/CCK interactions enhance satiation.
Rostral Ganglia Are Required for Induction But Not Expression of Crayfish Escape Reflex Habituation: Role of Higher Centers in Reprogramming Low-Level Circuits
Authors: Shirinyan David , Teshiba Terri , Taylor Karen , O'Neill Pia , Lee Sunhee Cho , Krasne Franklin B.
Published: December 2005

It is widely assumed that learning results from alterations in the strength of synapses within the neural pathways that mediate a learned behavioral response and that these alterations are directly caused by training-induced activity of neurons connected by the changing synapses. Initial evidence for this view came from studies of habituation of defensive reflexes in several invertebrate species. However, more recent studies of habituation of the escape reflex in one of these species, the crayfish, have shown that habituation is substantially caused by tonic inhibitory input from cephalic ganglia; this descending inhibition suppresses the activity of neurons within the escape circuit, which reside in caudal ganglia. Such control by descending inhibition indicates that animals with encephalized nervous systems do not entirely abdicate to low-level circuitry the important decision of whether to habituate to stimuli that might warn of danger. Higher centers in fact play a major role in controlling the habituation of this potentially life-saving protective response. Another way for higher centers to control lower ones would be to induce alteration of the lower center's intrinsic properties. Here, we show that, whereas descending input from higher ganglia is needed to induce habituation, once established, habituation persists even after rostral ganglia are disconnected. This provides evidence that lower-level neural circuits can be reprogrammed through transient interaction with higher ganglia to decrease their intrinsic tendency to produce escape.