bluuebunny/crossref_metadata_2025_split huggingface dataset:
Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes
Authors: Schaeffer Evelyne , Soros Vanessa B. , Greene Warner C. 
Published: January 2004

ABSTRACT

Virions of the type 1 human immunodeficiency virus (HIV-1) can enter target cells by fusion or endocytosis, with sharply different functional consequences. Fusion promotes productive infection of the target cell, while endocytosis generally leads to virion inactivation in acidified endosomes or degradation in lysosomes. Virion fusion and endocytosis occur equally in T cells, but these pathways have been regarded as independent because endocytosis of HIV virions requires neither CD4 nor CCR5/CXCR4 engagement in HeLa-CD4 cells. Using flow cytometric techniques to assess the binding and entry of green fluorescent protein (GFP)-Vpr-labeled HIV virions into primary peripheral blood mononuclear cells, we have found that HIV fusion and endocytosis are restricted to the CD4-expressing subset of cells and that both pathways commonly require the initial binding of HIV virions to surface CD4 receptors. Blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry via the endocytic pathway. Similarly, inhibition of endosome acidification with bafilomycin A1, concanamycin A, or NH
4
Cl enhanced entry via the fusion pathway. Although fusion remained dependent on CD4 and chemokine receptor binding, the endosome inhibitors did not alter surface expression of CD4 and CXCR4. These results suggest that fusion in the presence of the endosome inhibitors likely occurs within nonacidified endosomes. However, the ability of these inhibitors to impair vesicle trafficking from early to late endosomes in some cells could also increase the recycling of these virion-containing endosomes to the cell surface, where fusion occurs. In summary, our results reveal an unexpected, CD4-mediated reciprocal relationship between the pathways governing HIV virion fusion and endocytosis.
Multiple Modifications Allow High-Titer Production of Retroviral Vectors Carrying Heterologous Regulatory Elements
Authors: Hlavaty Juraj , Stracke Anika , Klein Dieter , Salmons Brian , Günzburg Walter H. , Renner Matthias 
Published: January 2004

ABSTRACT
Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3′ long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5′ and 3′ of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.
Replication of Poliovirus RNA with Complete Internal Ribosome Entry Site Deletions
Authors: Murray Kenneth E. , Steil Benjamin P. , Roberts Allan W. , Barton David J. 
Published: January 2004

ABSTRACT

cis
-acting RNA sequences and structures in the 5′ and 3′ nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5′ nontranslated region (NTR) has been implicated as a
cis
-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in
cis
for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.
Effects of Blocking Individual Maturation Cleavages in Murine Leukemia Virus Gag
Authors: Oshima Masamichi , Muriaux Delphine , Mirro Jane , Nagashima Kunio , Dryden Kelly , Yeager Mark , Rein Alan 
Published: January 2004

ABSTRACT
A single protein, termed Gag, is responsible for retrovirus particle assembly. After the assembled virion is released from the cell, Gag is cleaved at several sites by the viral protease (PR). The cleavages catalyzed by PR bring about a wide variety of physical changes in the particle, collectively termed maturation, and convert the particle into an infectious virion. In murine leukemia virus (MLV) maturation, Gag is cleaved at three sites, resulting in formation of the matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. We introduced mutations into MLV that inhibited cleavage at individual sites in Gag. All mutants had lost the intensely staining ring characteristic of immature particles; thus, no single cleavage event is required for this feature of maturation. Mutant virions in which MA was not cleaved from p12 were still infectious, with a specific infectivity only ∼10-fold below that of the wild type. Particles in which p12 and CA could not be separated from each other were noninfectious and lacked a well-delineated core despite the presence of dense material in their interiors. In both of these mutants, the dimeric viral RNA had undergone the stabilization normally associated with maturation, suggesting that this change may depend upon the separation of CA from NC. Alteration of the C-terminal end of CA blocked CA-NC cleavage but also reduced the efficiency of particle formation and, in some cases, severely disrupted the ability of Gag to assemble into regular structures. This observation highlights the critical role of this region of Gag in assembly.
Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors Pseudotyped with Envelope Glycoproteins Derived from Ross River Virus and Semliki Forest Virus
Authors: Kahl Christoph A. , Marsh Jon , Fyffe Joanne , Sanders David A. , Cornetta Kenneth 
Published: January 2004

ABSTRACT
Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors.
Enterovirus Capsid Interactions with Decay-Accelerating Factor Mediate Lytic Cell Infection
Authors: Newcombe Nicole G. , Johansson E. Susanne , Au Gough , Lindberg A. Michael , Barry Richard D. , Shafren Darren R. 
Published: January 2004

ABSTRACTThe cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.
A Novel Viral Transcript with Homology to Human Interleukin-10 Is Expressed during Latent Human Cytomegalovirus Infection
Authors: Jenkins Christina , Abendroth Allison , Slobedman Barry 
Published: January 2004

ABSTRACT
Human cytomegalovirus (CMV) establishes latent infections in hematopoietic cells such as granulocyte-macrophage progenitors (GM-Ps). During latency the virus is sequestered in a nonreplicating state, although limited transcriptional activity has been previously reported. In this study we sought to further examine viral gene expression during the latent phase of infection. Using an experimental model of latency, primary human GM-Ps were latently infected with CMV strain Toledo and extracted RNA subjected to reverse transcription-PCR by using CMV gene-specific primers. Using this approach, we detected transcription from the UL111.5A region of the viral genome. This transcription was also detected in GM-Ps latently infected with AD169 and Towne strains, indicating that expression was CMV strain independent. Significantly, we detected UL111.5A-region transcripts in mononuclear cells from healthy bone marrow and mobilized peripheral blood allograft donors, demonstrating expression during natural latent infection. Mapping experiments with RNA extracted from latently infected GM-Ps revealed the expression of a novel UL111.5A region transcript with a splicing pattern that differed from that reported during productive infection of permissive cells. This UL111.5A region transcript expressed during latent infection is predicted to encode a 139-amino-acid protein with homology to the potent immunosuppressor interleukin-10 (IL-10) and to the viral IL-10 homolog that is expressed during productive CMV infection. Expression of a latency-associated cmvIL-10 may confer upon the virus an ability to avoid immune recognition and clearance during the latent phase of infection.
CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection
Authors: Zhang Jie , Randall Glenn , Higginbottom Adrian , Monk Peter , Rice Charles M. , McKeating Jane A. 
Published: January 2004

ABSTRACT
CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.
Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System
Authors: Xie Baode , Calabro Valerie , Wainberg Mark A. , Frankel Alan D. 
Published: January 2004

ABSTRACT
The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a “chameleon,” adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.
Dysregulation of the Polo-Like Kinase Pathway in CD4
+
T Cells Is Characteristic of Pathogenic Simian Immunodeficiency Virus Infection
Authors: Bostik Pavel , Dodd Geraldine L. , Villinger Francois , Mayne Ann E. , Ansari Aftab A. 
Published: January 2004

ABSTRACT

CD4
+
T-cell dysfunction highlighted by defects within the intracellular signaling cascade and cell cycle has long been characterized as a direct and/or indirect consequence of human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM). Dysregulation of the M phase of the cell cycle is a well-documented effect of HIV or SIV infection both in vivo and in vitro. In this study the effect of SIV infection on the modulation of two important regulators of the M phase—polo-like kinases Plk3 and Plk1—was investigated. We have previously shown that Plk3 is markedly downregulated in CD4
+
T cells from SIV-infected disease-susceptible RM but not SIV-infected disease-resistant sooty mangabeys (SM), denoting an association of downregulation with disease progression. Here we show that, in addition to the downregulation, Plk3 exhibits aberrant activation patterns in the CD4
+
T cells from SIV-infected RM following T-cell receptor stimulation. Interestingly, in vitro SIV infection of CD4
+
T cells leads to the upregulation, rather than downregulation, of Plk3, suggesting that different mechanisms operate in vitro and in vivo. In addition, CD4
+
T cells from RM with high viral loads exhibited consistent and significant upregulation of Plk1, concurrent with an aberrant activation-induced Plk1 response, suggesting complex mechanisms of SIV-induced M-phase abnormalities in vivo. Altogether this study presents a novel mechanism underlying M-phase defects observed in CD4
+
T cells from HIV or SIV-infected disease-susceptible humans and RM which may contribute to aberrant T-cell responses and disease pathogenesis.
Systemic Immune Deficiency Necessary for Cytomegalovirus Invasion of the Mature Brain
Authors: Reuter Jon D. , Gomez Daniel L. , Wilson Jean H. , van den Pol Anthony N. 
Published: January 2004

ABSTRACT

Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45
+
) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.
Both the PPPY and PTAP Motifs Are Involved in Human T-Cell Leukemia Virus Type 1 Particle Release
Authors: Wang Huating , Machesky Nicholas J. , Mansky Louis M. 
Published: January 2004

ABSTRACT
In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.