bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an Essential Molecular Contact Point on the Duck Hepatitis B Virus Reverse Transcriptase
Authors: Cao Feng , Badtke Matthew P. , Metzger Lisa M. , Yao Ermei , Adeyemo Babatunde , Gong Yunhao , Tavis John E. 
Published: July 2005

ABSTRACT
The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.
Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections
Authors: Guerrero-Plata Antonieta , Baron Samuel , Poast Joyce S. , Adegboyega Patrick A. , Casola Antonella , Garofalo Roberto P. 
Published: July 2005

ABSTRACT

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the
Paramyxoviridae
family,
Pneumovirinae
subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-α/β) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-α in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-α, or directly with recombinant IFN-α showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-α than hMPV. Similarly, when the ability to directly induce IFN-α production was assessed, RSV was clearly a weaker inducer of IFN-α than hMPV, as shown by both kinetics and the absolute amount of IFN-α secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-α production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-α production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-α in the lung during infection by members of the
Pneumovirinae
family.
Formaldehyde-Treated, Heat-Inactivated Virions with Increased Human Immunodeficiency Virus Type 1 Env Can Be Used To Induce High-Titer Neutralizing Antibody Responses
Authors: Poon B. , Hsu J. F. , Gudeman V. , Chen I. S. Y. , Grovit-Ferbas K. 
Published: July 2005

ABSTRACT
The lack of success of subunit human immunodeficiency virus (HIV) type 1 vaccines to date suggests that multiple components or a complex virion structure may be required. We hypothesized that the failure of current vaccine strategies to induce protective antibodies is linked to the inability of native envelope structures to readily elicit these types of antibodies. We have previously reported on the ability of a formaldehyde-treated, heat-inactivated vaccine to induce modest antibody responses in animal vaccine models. We investigated here whether immunization for HIV with an envelope-modified, formaldehyde-stabilized, heat-inactivated virion vaccine could produce higher-titer and/or broader neutralizing antibody responses. Thus, a clade B vaccine which contains a single point mutation in gp41 (Y706C) that results in increased incorporation of oligomeric Env into virions was constructed. This vaccine was capable of inducing high-titer antibodies that could neutralize heterologous viruses, including those of clades A and C. These results further support the development of HIV vaccines with modifications in native Env structures for the induction of neutralizing antibody responses.
Encephalomyocarditis Virus Induces PKR-Independent Mitogen-Activated Protein Kinase Activation in Macrophages
Authors: Moran Jason M. , Moxley Michael A. , Buller R. Mark L. , Corbett John A. 
Published: July 2005

ABSTRACTIn this study, we provide evidence that the double-stranded RNA-dependent protein kinase (PKR) is not required for virus-induced expression of inducible nitric oxide synthase (iNOS) or the activation of specific signaling pathways in macrophages. The infection of RAW264.7 cells with encephalomyocarditis virus (EMCV) induces iNOS expression and nitric oxide production, which are unaffected by a dominant-negative mutant of PKR. EMCV infection also activates the mitogen-activated protein kinase, cyclic AMP response element binding protein, and nuclear factor κB (NF-κB) signaling cascades at 15 to 30 min postinfection in PKR+/+and PKR−/−macrophages. Activation of these signaling cascades does not temporally correlate with PKR activity or the accumulation of EMCV RNA, suggesting that an interaction between a structural component of the virion and the cell surface may activate macrophages. Consistent with this hypothesis, empty EMCV capsids induced comparable levels of iNOS expression, nitrite production, and activation of these signaling cascades to those induced by intact virions. These findings support the hypothesis that virion-host cell interactions are primary mediators of the PKR-independent activation of signaling pathways that participate in the macrophage antiviral response of inflammatory gene expression.
Determinants of Human Immunodeficiency Virus Type 1 Resistance to Membrane-Anchored gp41-Derived Peptides
Authors: Lohrengel Sabine , Hermann Felix , Hagmann Isabel , Oberwinkler Heike , Scrivano Laura , Hoffmann Caroline , von Laer Dorothee , Dittmar Matthias T. 
Published: July 2005

ABSTRACTThe expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol.75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1NL4-3and HIV-1BaLby serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1NL4-3a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1BaLthe majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1BaLresults in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1NL4-3did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol.78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.
Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å
Authors: Xu Ting , Sampath Aruna , Chao Alex , Wen Daying , Nanao Max , Chene Patrick , Vasudevan Subhash G. , Lescar Julien 
Published: July 2005

ABSTRACT

Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the
Flaviviridae
replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3′ single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.
Ebola Virus VP40 Late Domains Are Not Essential for Viral Replication in Cell Culture
Authors: Neumann Gabriele , Ebihara Hideki , Takada Ayato , Noda Takeshi , Kobasa Darwyn , Jasenosky Luke D. , Watanabe Shinji , Kim Jin H. , Feldmann Heinz , Kawaoka Yoshihiro 
Published: July 2005

ABSTRACT
Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.
Gamma Interferon Can Block Herpes Simplex Virus Type 1 Reactivation from Latency, Even in the Presence of Late Gene Expression
Authors: Decman Vilma , Kinchington Paul R. , Harvey Stephen A. K. , Hendricks Robert L. 
Published: July 2005

ABSTRACTHerpes simplex virus type 1 (HSV-1)-specific CD8+T cells and the cytokine gamma interferon (IFN-γ) are persistently present in trigeminal ganglia (TG) harboring latent HSV-1. We define “latency” as the retention of functional viral genomes in sensory neurons without the production of infectious virions and “reactivation” as a multistep process leading from latency to virion assembly. CD8+T cells can block HSV-1 reactivation in ex vivo mouse TG cultures and appear to be the sole source of IFN-γ in these cultures. Here we demonstrate that IFN-γ alone can block HSV-1 reactivation in some latently infected neurons, and we identify points of intervention in the life cycle of the reactivating virus. Cell suspensions of TG that were latently infected with recombinant RE HSV-1 expressing enhanced green fluorescent protein from the promoter for infected cell protein 0 (ICP0) or glycoprotein C (gC) were depleted of endogenous CD8+or CD45+cells and cultured in the presence or absence of IFN-γ. Our results demonstrate that IFN-γ acts on latently infected neurons to inhibit (i) HSV-1 reactivation, (ii) ICP0 promoter activity, (iii) gC promoter activity, and (iv) reactivation in neurons in which the ICP0 or gC promoter is active. Interestingly, we detected transcripts for ICP0, ICP4, and gH in neurons that expressed the ICP0 promoter but were prevented by IFN-γ from reactivation and virion formation. Thus, the IFN-γ blockade of HSV-1 reactivation from latency in neurons is associated with an inhibition of the expression of the ICP0 gene (required for reactivation) and a blockade of a step that occurs after the expression of at least some viral structural genes.
Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model
Authors: Mori Kazuyasu , Sugimoto Chie , Ohgimoto Shinji , Nakayama Emi E. , Shioda Tatsuo , Kusagawa Shigeru , Takebe Yutaka , Kano Munehide , Matano Tetsuro , Yuasa Takae , Kitaguchi Daisuke , Miyazawa Masaaki , Takahashi Yumiko , Yasunami Michio , Kimura Akinori , Yamamoto Naoki , Suzuki Yasuo , Nagai Yoshiyuki 
Published: July 2005

ABSTRACT
The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (Δ5G). In Δ5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and Δ5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in Δ5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in Δ5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.
Intrinsic Functional Dysregulation of CD4 T Cells Occurs Rapidly following Persistent Viral Infection
Authors: Brooks David G. , Teyton Luc , Oldstone Michael B. A. , McGavern Dorian B. 
Published: July 2005

ABSTRACTEffective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.
Association of Bovine Papillomavirus E2 Protein with Nuclear Structures In Vivo
Authors: Kurg Reet , Sild Kristiina , Ilves Aigi , Sepp Mari , Ustav Mart 
Published: July 2005

ABSTRACT
Papillomaviruses are small DNA viruses which have the capacity to establish a persistent infection in mammalian epithelial cells. The papillomavirus E2 protein is a central coordinator of viral gene expression, genome replication, and maintenance. We have investigated the distribution of bovine papillomavirus E2 protein in nuclei of proliferating cells and found that E2 is associated with cellular chromatin. This distribution does not change during the entire cell cycle. The N-terminal transactivation domain, but not the C-terminal DNA-binding domain, of the E2 protein is responsible for this association. The majority of the full-length E2 protein can only be detected in chromatin-enriched fractions but not as a free protein in the nucleus. Limited micrococcal nuclease digestion revealed that the E2 protein partitioned to different chromatin regions. A fraction of the E2 protein was located at nuclear sites that are resistant against nuclease attack, whereas the remaining E2 resided on compact chromatin accessible to micrococcal nuclease. These data suggest that there are two pools of E2 in the cell nucleus: one that localizes on transcriptionally inactive compact chromatin and the other, which compartmentalizes to transcriptionally active nuclear structures of the cell. Our data also suggest that E2 associates with chromatin through cellular protein(s), which in turn is released from chromatin at 0.4 M salt.
Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly
Authors: Newcomb William W. , Homa Fred L. , Brown Jay C. 
Published: July 2005

ABSTRACT
DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.
The
Lymantria dispar
Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus
Authors: Slavicek James M. , Popham Holly J. R. 
Published: July 2005

ABSTRACT

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two
enhancin
genes (E1 and E2) in the
Lymantria dispar
multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV
enhancin
genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of ∼84 kDa and 90 kDa, respectively, on Western blots of extracts from
L. dispar
652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the
enhancin
genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.