bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adaptive Mutations in the V3 Loop of gp120 Enhance Fusogenicity of Human Immunodeficiency Virus Type 1 and Enable Use of a CCR5 Coreceptor That Lacks the Amino-Terminal Sulfated Region
Authors: Platt Emily J. , Kuhmann Shawn E. , Rose Patrick P. , Kabat David 
Published: July 2002

ABSTRACT
To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.
Glycosphingolipid Binding Specificities of Rotavirus: Identification of a Sialic Acid-Binding Epitope
Authors: Delorme Cécile , Brüssow Harald , Sidoti Josette , Roche Niamh , Karlsson Karl-Anders , Neeser Jean-Richard , Teneberg Susann 
Published: July 2002

ABSTRACT
The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcα3-Galβ) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcα3Galβ is preferred to NeuAcα3Galβ. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.
Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro
Authors: Yoon Miran , Smith Deborah H. , Ward Peter , Medrano Francisco J. , Aggarwal Aneel K. , Linden R. Michael 
Published: July 2002

ABSTRACT

The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (
AAVS1
) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,
REP
, with sequences present in
AAVS1
that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.
AlaArg Motif in the Carboxyl Terminus of the γ
1
34.5 Protein of Herpes Simplex Virus Type 1 Is Required for the Formation of a High-Molecular-Weight Complex That Dephosphorylates eIF-2α
Authors: Cheng Guofeng , Gross Martin , Brett Marie-Elena , He Bin 
Published: July 2002

ABSTRACT

The γ
1
34.5 protein of herpes simplex virus (HSV) type 1 functions to prevent the shutoff of protein synthesis mediated by the double-stranded-RNA-dependent protein kinase PKR. This is because γ
1
34.5 associates with protein phosphatase 1 (PP1) through its carboxyl terminus, forming a high-molecular-weight complex that dephosphorylates the α subunit of translation initiation factor eIF-2 (eIF-2α). Here we show that Val
193
Glu and Phe
195
Leu substitutions in the PP1 signature motif of the γ
1
34.5 protein abolished its ability to redirect PP1 to dephosphorylate eIF-2α and replication of mutant viruses was severely impaired. The γ
1
34.5 protein, when expressed in Sf9 cells using a recombinant baculovirus, was capable of directing specific eIF-2α dephosphorylation. Deletions of amino acids 258 to 263 had no effect on activity of γ
1
34.5. However, deletions of amino acids 238 to 258 abolished eIF-2α phosphatase activity but not PP1 binding activity. Interestingly, deletions in the AlaArg motif of the carboxyl terminus disrupted the high-molecular-weight complex that is required for dephosphorylation of eIF-2α. These results demonstrate that γ
1
34.5 is functionally active in the absence of any other HSV proteins. In addition to a PP1 binding domain, the carboxyl terminus of γ
1
34.5 contains an effector domain that is required to form a functional complex.
Comparison of Cell Cycle Arrest, Transactivation, and Apoptosis Induced by the Simian Immunodeficiency Virus SIVagm and Human Immunodeficiency Virus Type 1vprGenes
Authors: Zhu Yonghong , Gelbard Harris A. , Roshal Mikhail , Pursell Shannon , Jamieson Beth D. , Planelles Vicente 
Published: July 2002

ABSTRACTAll primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1)vprgene. HIV-1vprencodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains avprhomologue, which encodes a 118-amino-acid protein. SIVagmvpris structurally and functionally related to HIV-1vpr. The present study focuses on how three specific functions (transactivation, induction of G2arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagmvpr. While our study supports previous reports demonstrating a causal relationship between induction of G2arrest and transactivation for HIV-1vpr, we demonstrate that the same is not true for SIVagmvpr. Transactivation by SIVagmvpris independent of cell cycle perturbation. In addition, we show that induction of G2arrest is necessary for the induction of apoptosis by HIV-1vprbut that the induction of apoptosis by SIVagmvpris cell cycle independent. Finally, while SIVagmvprretains its transactivation function in human cells, it is unable to induce G2arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagmvprare species specific. Taken together, our results suggest that while the multiple functions ofvprare conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.
Cross-Presentation of Human Cytomegalovirus pp65 (UL83) to CD8+T Cells Is Regulated by Virus-Induced, Soluble-Mediator-Dependent Maturation of Dendritic Cells
Authors: Arrode Géraldine , Boccaccio Claire , Abastado Jean-Pierre , Davrinche Christian 
Published: July 2002

ABSTRACTCytotoxic CD8+T lymphocytes (CTL) directed against the matrix protein pp65 are major effectors in controlling infection against human cytomegalovirus (HCMV), a persistent virus of theBetaherpesvirusfamily. We previously suggested that cross-presentation of pp65 by nonpermissive dendritic cells (DCs) could overcome viral strategies that interfere with activation of CTL (G. Arrode, C. Boccaccio, J. Lule, S. Allart, N. Moinard, J. Abastado, A. Alam, and C. Davrinche, J. Virol. 74:10018–10024, 2000). It is well established that mature DCs are very potent in initiating T-cell-mediated immunity. Consequently, the DC maturation process is a key step targeted by viruses in order to avoid an immune response. Here, we report that immature DCs maintained in coculture with infected human (MRC5) fibroblasts acquired pp65 from early-infected cells for cross-presentation to specific HLA-A2-restricted CTL. In contrast, coculture of DCs in the presence of late-infected cells decreased their capacity to stimulate CTL. Analyses of DC maturation after either coculture with infected MRC5 cells or incubation with infected-cell-conditioned medium revealed that acquisition of a mature phenotype was a prerequisite for efficient stimulation of CTL and that soluble factors secreted by infected cells were responsible for both up and down regulation of CD83 expression on DCs. We identified transforming growth factor β1 secreted by late HCMV-infected cells as one of these down regulating mediators. These findings suggest that HCMV has devised another means to compromise immune surveillance mechanisms. Together, our data indicate that recognition of HCMV-infected cells by DCs has to occur early after infection to avoid immune evasion and to allow generation of anti-HCMV CTL.
RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication
Authors: Brincat Jennifer L. , Pfeiffer Julie K. , Telesnitsky Alice 
Published: July 2002

ABSTRACT
It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.
Herpes Simplex Virus Tegument Protein VP22 Contains Overlapping Domains for Cytoplasmic Localization, Microtubule Interaction, and Chromatin Binding
Authors: Martin Ana , O'Hare Peter , McLauchlan John , Elliott Gillian 
Published: July 2002

ABSTRACT
We have previously shown that the 301-amino-acid herpes simplex virus tegument protein VP22 exhibits a range of subcellular localization patterns when expressed in isolation from other virus proteins. By using live-cell analysis of cells expressing green fluorescent protein (GFP)-tagged VP22 we have shown that when VP22 is first expressed in the cell it localizes to the cytoplasm, where, when present at high enough concentrations, it can assemble onto microtubules, causing them to bundle and become highly stabilized. In addition we have shown that when a cell expressing VP22 enters mitosis, the cytoplasmic population of VP22 translocates to the nucleus, where it efficiently binds mitotic chromatin. Here we have investigated the specific regions of the VP22 open reading frame required for these properties. Using GFP-VP22 as our starting molecule, we have constructed a range of N- and C-terminal truncations and analyzed their localization patterns in live cells. We show that the C-terminal 242 residues of VP22 are sufficient to induce microtubule bundling. Within this subregion, the C-terminal 89 residues contain a signal for cytoplasmic localization of the protein, while a larger region comprising the C-terminal 128 residues of the VP22 protein is required for mitotic chromatin binding. Furthermore, a central 100-residue domain of VP22 maintains the ability to bind microtubules without inducing bundling, suggesting that additional regions flanking this microtubule binding domain may be required to alter the microtubule network. Hence, the signals involved in dictating the complex localization patterns of VP22 are present in overlapping regions of the protein.
Human TATA Binding Protein Inhibits Human Papillomavirus Type 11 DNA Replication by Antagonizing E1-E2 Protein Complex Formation on the Viral Origin of Replication
Authors: Hartley Kelly A. , Alexander Kenneth A. 
Published: July 2002

ABSTRACT

The human papillomavirus (HPV) protein E2 possesses dual roles in the viral life cycle. By interacting directly with host transcription factors in basal keratinocytes, E2 promotes viral transcription. As keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, to activate vegetative viral DNA replication. How E2's major role switches from transcription to replication during keratinocyte differentiation is not understood, but the presence of a TATA site near the viral origin of replication led us to hypothesize that TATA-binding protein (TBP) could affect HPV replication. Here we show that the C-terminal domain of TBP (TBPc) is a potent inhibitor of E2-stimulated HPV DNA replication in vitro (50% inhibitory concentration = 0.56 nM). Increasing the E1 concentration could not overcome TBPc inhibition in replication assays, indicating that TBPc is a noncompetitive inhibitor of E1 binding. While direct E2-TBPc association could be demonstrated, this interaction could not fully account for the mechanism of TBPc-mediated inhibition of viral replication. Because E2 supports sequence-specific binding of E1 to the viral
ori
, we proposed that TBPc antagonizes E1-
ori
association indirectly through inhibition of E2-DNA binding. Indeed, TBPc potently antagonized E2 binding to DNA in the absence (

K
i

= 0.5 ± 0.1 nM) and presence (

K
i

= 0.6 ± 0.3 nM) of E1. Since E2 and TBPc cannot be coadjacent on viral sequences, direct DNA-binding competition between TBPc and E2 was responsible for replication inhibition. Given the ability of TBPc to inhibit HPV DNA replication in vitro and data indicating that TBPc antagonized E2-
ori
association, we propose that transcription factors regulate HPV DNA replication as well as viral transcription.
The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication
Authors: Pathakamuri Joseph A. , Theilmann David A. 
Published: July 2002

ABSTRACT

IE1 is a potent transcriptional transactivator of the baculovirus
Orgyia pseudotsugata
multiple nucleopolyhedrovirus (Op
M
NPV) and has been shown to be essential for viral DNA replication. IE1 contains an acidic activation domain (AAD) at the N terminus that is essential for transcriptional transactivation, but its role in viral DNA replication is unknown. In this study the role of the IE1 AAD in DNA replication is investigated. We have determined that deletion of the AAD eliminates the ability of IE1 to support DNA replication, showing that the AAD is essential for DNA replication as well as transcriptional transactivation. Replacement of the AAD with the archetype domain from herpesvirus VP16 and the evolutionarily related domain from
Autographa californica M
NPV (Ac
M
NPV) IE1 produces chimeric proteins that are potent transactivators. Surprisingly, however, these chimeric proteins were unable to support DNA replication, indicating that there is a host- or virus-specific replication subdomain in the AAD that was not functionally replaced by the VP16 or Ac
M
NPV AAD. Using N- and C-terminal deletion mutants, the region of the AAD that was essential for DNA replication was mapped to amino acids 1 to 65. AAD deletion mutants also showed that an IE1 that is functional for transcriptional transactivation is not required for viral DNA replication. The IE1 AAD therefore contains an essential replication domain that is separable from the transcriptional activation domains. Our results suggest that IE1 specifically interacts with a component of the viral replication complex, supporting the view that it acts as a nucleating factor by binding to the viral replication origins.
Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat
Authors: Tumpey Terrence M. , Suarez David L. , Perkins Laura E. L. , Senne Dennis A. , Lee Jae-gil , Lee Youn-Jeong , Mo In-Pil , Sung Haan-Woo , Swayne David E. 
Published: July 2002

ABSTRACT
Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.
The Majority of Infiltrating CD8+T Cells in the Central Nervous System of Susceptible SJL/J Mice Infected with Theiler's Virus Are Virus Specific and Fully Functional
Authors: Kang Bong-Su , Lyman Michael A. , Kim Byung S. 
Published: July 2002

ABSTRACTTheiler's virus infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains, such as SJL/J, and serves as a relevant infectious model for human multiple sclerosis. It has been previously suggested that susceptible SJL/J mice do not mount an efficient cytotoxic T-lymphocyte (CTL) response to the virus. In addition, genetic studies have shown that resistance to Theiler's virus-induced demyelinating disease is linked to theH-2Dmajor histocompatibility complex class I locus, suggesting that a compromised CTL response may contribute to the susceptibility of SJL/J mice. Here we show that SJL/J mice do, in fact, generate a CD8+T-cell response in the CNS that is directed against one dominant (VP3159-166) and two subdominant (VP111-20and VP3173-181) capsid protein epitopes. These virus-specific CD8+T cells produce gamma interferon (IFN-γ) and lyse target cells in the presence of the epitope peptides, indicating that these CNS-infiltrating CD8+T cells are fully functional effector cells. Intracellular IFN-γ staining analysis indicates that greater than 50% of CNS-infiltrating CD8+T cells are specific for these viral epitopes at 7 days postinfection. Therefore, the susceptibility of SJL/J mice is not due to the lack of an early functional Theiler's murine encephalomyelitis virus-specific CTL response. Interestingly, T-cell responses to all three epitopes are restricted by the H-2Ksmolecule, and this skewed class I restriction may be associated with susceptibility to demyelinating disease.