bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Synthetic Immunomodulator Murabutide Controls Human Immunodeficiency Virus Type 1 Replication at Multiple Levels in Macrophages and Dendritic Cells
Authors: Darcissac Edith C. A. , Truong Marie-José , Dewulf Joëlle , Mouton Yves , Capron André , Bahr George M. 
Published: July 2002

ABSTRACTMacrophages and dendritic cells are known to play an important role in the establishment and persistence of human immunodeficiency virus (HIV) infection. Besides antiretroviral therapy, several immune-based interventions are being evaluated with the aim of achieving better control of virus replication in reservoir cells. Murabutide is a safe synthetic immunomodulator presenting a capacity to enhance nonspecific resistance against viral infections and to target cells of the reticuloendothelial system. In this study, we have examined the ability of Murabutide to control HIV type 1 (HIV-1) replication in acutely infected monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Highly significant suppression of viral replication was consistently observed in Murabutide-treated cultures of both cell types. Murabutide did not affect virus entry, reverse transcriptase activity, or early proviral DNA formation in the cytoplasm of infected cells. However, treated MDMs and MDDCs showed a dramatic reduction in nuclear viral two-long terminal repeat circular form and viral mRNA transcripts. This HIV-1-suppressive activity was not mediated by inhibiting cellular DNA synthesis or by activating p38 mitogen-activated protein kinase. Furthermore, Murabutide-stimulated cells expressed reduced CD4 and CCR5 receptors and secreted high levels of β-chemokines, although neutralization of the released chemokines did not alter the HIV-1-suppressive activity of Murabutide. These results provide evidence that a clinically acceptable immunomodulator can activate multiple effector pathways in macrophages and in dendritic cells, rendering them nonpermissive for HIV-1 replication.
Mutations in the 5′ Nontranslated Region of Bovine Viral Diarrhea Virus Result in Altered Growth Characteristics
Authors: Becher Paul , Orlich Michaela , Thiel Heinz-Jürgen 
Published: July 2002

ABSTRACT

The 5′ nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional
cis
-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5′-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5′ NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5′-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5′-terminal sequence; (iii) provided the conserved sequence motif 5′-GUAU was retained at the 5′ end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5′ NTR mutants correlated with reduced amounts of accumulated viral RNAs.
Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region
Authors: García-Escudero Ramón , Viñuela Eladio 
Published: July 2002

ABSTRACT

A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show that
cis
-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.
Study of Full-Length Porcine Endogenous Retrovirus Genomes with Envelope Gene Polymorphism in a Specific-Pathogen-Free Large White Swine Herd
Authors: Bösch Steffi , Arnauld Claire , Jestin André 
Published: July 2002

ABSTRACT

Specific-pathogen-free (SPF) swine appear to be the most appropriate candidate for pig to human xenotransplantation. Still, the risk of endogenous retrovirus transmission represents a major obstacle, since two human-tropic porcine endogenous retroviruses (PERVs) had been characterized in vitro (P. Le Tissier, J. P. Stoye, Y. Takeuchi, C. Patience, and R. A. Weiss, Nature 389:681–682, 1997). Here we addressed the question of PERV distribution in a French Large White SPF pig herd in vivo. First, PCR screening for previously described PERV envelope genes
envA
,
envB
, and
envC
(D. E. Akiyoshi, M. Denaro, H. Zhu, J. L. Greenstein, P. Banerjee, and J. A. Fishman, J. Virol. 72:4503–4507, 1998; Le Tissier et al., op. cit.). demonstrated ubiquity of
envA
and
envB
sequences, whereas
envC
genes were absent in some animals. On this basis, selective out-breeding of pigs of remote origin might be a means to reduce proviral load in organ donors. Second, we investigated PERV genome carriage in
envC
negative swine. Eleven distinct full-length PERV transcripts were isolated. The sequence of the complete envelope open reading frame was determined. The deduced amino acid sequences revealed the existence of four clones with functional and five clones with defective PERV PK-15 A- and B-like envelope sequences. The occurrence of easily detectable levels of PERV variants in different pig tissues in vivo heightens the need to assess PERV transmission in xenotransplantation animal models.
RNase L-Independent Specific 28S rRNA Cleavage in Murine Coronavirus-Infected Cells
Authors: Banerjee Sangeeta , An Sungwhan , Zhou Aimin , Silverman Robert H. , Makino Shinji 
Published: July 2002

ABSTRACT
We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.
Leader Sequences Downstream of the Primer Binding Site Are Important for Efficient Replication of Simian Immunodeficiency Virus
Authors: Guan Yongjun , Whitney James B. , Diallo Karidia , Wainberg Mark A. 
Published: July 2002

ABSTRACT
Simian immunodeficiency virus (SIV) infection of macaques is remarkably similar to that of human immunodeficiency virus type 1 (HIV-1) in humans, and the SIV-macaque system is a good model for AIDS research. We have constructed an SIV proviral DNA clone that is deleted of 97 nucleotides (nt), i.e., construct SD, at positions (+322 to +418) immediately downstream of the primer binding site (PBS) of SIVmac239. When this construct was transfected into COS-7 cells, the resultant viral progeny were severely impaired with regard to their ability to replicate in C8166 cells. Further deletion analysis showed that a virus termed SD1, containing a deletion of 23 nt (+322 to +344), was able to replicate with wild-type kinetics, while viruses containing deletions of 21 nt (+398 to +418) (construct SD2) or 53 nt (+345 to +397) (construct SD3) displayed diminished capacity in this regard. Both the SD2 and SD3 viruses were also impaired with regard to ability to package viral RNA, while SD1 viruses were not. The SD and SD3 constructs did not revert to increased replication ability in C8166 cells over 6 months in culture. In contrast, long-term passage of the SD2 mutated virus resulted in a restoration of replication capacity, due to the appearance of four separate point mutations. Two of these substitutions were located in leader sequences of viral RNA within the PBS and the dimerization initiation site (DIS), while the other two were located within two distinct Gag proteins, i.e., CA and p6. The biological relevance of three of these point mutations was confirmed by site-directed mutagenesis studies that showed that SD2 viruses containing each of these substitutions had regained a significant degree of viral replication capacity. Thus, leader sequences downstream of the PBS, especially the U5-leader stem and the DIS stem-loop, are important for SIV replication and for packaging of the viral genome.
Replicon Vectors Derived from Sindbis Virus and Semliki Forest Virus That Establish Persistent Replication in Host Cells
Authors: Perri Silvia , Driver David A. , Gardner Jason P. , Sherrill Scott , Belli Barbara A. , Dubensky Thomas W. , Polo John M. 
Published: July 2002

ABSTRACT
Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.
Sequestration of TT Virus of Restricted Genotypes in Peripheral Blood Mononuclear Cells
Authors: Okamoto Hiroaki , Takahashi Masaharu , Kato Naomi , Fukuda Masako , Tawara Akio , Fukuda Satoko , Tanaka Takeshi , Miyakawa Yuzo , Mayumi Makoto 
Published: July 2002

ABSTRACT
Peripheral blood mononuclear cells (PBMC) harbored TT virus (TTV) of genotypes (3 and 4) different from those (1 and 2) of free virions in plasma of the same individuals. PBMC may act as a reservoir, and TTV of particular genotypes might have tropism for hematopoietic cells.
Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity
Authors: Halvas Elias K. , Svarovskaia Evguenia S. , Pathak Vinay K. 
Published: July 2002

ABSTRACT

Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (
lacZ
), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.
Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains
Authors: Chan Szeman Ruby , Chandran Bala 
Published: July 2002

ABSTRACT
Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118–126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.
Epstein-Barr Virus Immediate-Early Proteins BZLF1 and BRLF1 Activate the ATF2 Transcription Factor by Increasing the Levels of Phosphorylated p38 and c-Jun N-Terminal Kinases
Authors: Adamson Amy L. , Darr Dayle , Holley-Guthrie Elizabeth , Johnson Robert A. , Mauser Amy , Swenson Jennifer , Kenney Shannon 
Published: July 2002

ABSTRACT
Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.
Alpha/Beta Interferon Protects Adult Mice from Fatal Sindbis Virus Infection and Is an Important Determinant of Cell and Tissue Tropism
Authors: Ryman Kate D. , Klimstra William B. , Nguyen Khuong B. , Biron Christine A. , Johnston Robert E. 
Published: July 2002

ABSTRACTInfection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-α/βR−/−) mice, we have assessed the contribution of IFN-α/β in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-α/βR−/−mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-γ, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-α/β-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-α/βR−/−mice. Thus, IFN-α/β protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-α/β system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.
Mechanisms of Human Papillomavirus E2-Mediated Repression of Viral Oncogene Expression and Cervical Cancer Cell Growth Inhibition
Authors: Nishimura Akiko , Ono Takeshi , Ishimoto Akinori , Dowhanick Jennifer J. , Frizzell Margaret A. , Howley Peter M. , Sakai Hiroyuki 
Published: July 2002

ABSTRACT

The papillomavirus
E2
gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viral
E6/E7
promoter. It had been previously postulated that transcriptional repression of the
E6/E7
promoter results from E2 binding its cognate sites proximal to the
E6/E7
promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates.