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Results for “viral complexity”

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At least 667 records · Page 37Linked to original sources

PubMed1995

HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator.

The HIV-1 matrix (MA) protein contains two subcellular localization signals with opposing effects. A myristoylated N-terminus governs particle assembly at the plasma membrane, and a nucleophilic motif facilitates import of the viral preintegration complex into the nucleus of nondividing cells. Here, we show that myristoylation acts as the MA dominant targeting signal in HIV-1 producer cells. During virus assembly, a subset of MA is phosphorylated on the C-terminal tyrosine by a virion-associated cellular protein kinase. Tyrosine-phosphorylated MA is then preferentially transported to the nucleus of target cells. An MA tyrosine mutant virus grows normally in dividing cells, but is blocked for nuclear import in terminally differentiated macrophages. MA tyrosine phosphorylation thus reveals the karyophilic properties of this protein within the HIV-1 preintegration complex, thereby playing a critical role for infection of nondividing cells.

Amino Acid Sequence↗

PubMed2006

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines.

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.

Calcium↗

PubMed1985

Subunit vaccines against exogenous retroviruses: overview and perspectives.

Vaccines prepared from purified viral envelope complexes are effective against certain animal model tumors induced by exogenous retroviruses. Related viruses have recently been isolated from humans and obviously cause adult T-cell leukemia and the acquired immunodeficiency syndrome. Knowledge accumulated in experiments with subunit vaccines against animal retroviruses could help to develop immunopreventive regimens against human retroviruses.

Acquired Immunodeficiency Syndrome↗

PubMed1979

Distinctive RNA transcriptase, polyadenylic acid polymerase, and polyuridylic acid polymerase activities associated with Pichinde virus.

Three RNA polymerase activities were found and associated with purified Pichinde virus, a member of the Arenaviridae. A heat-labile polymerase activity which required all four ribonucleoside triphosphates for optimal activity co-sedimented on sucrose gradient centrifugation with the viral ribonucleoprotein complex from detergent-disrupted virus preparations. This enzyme synthesized heteropolymers which represented about 23% of the genome RNA as determined by nucleic acid hybridization. Two relatively heat-stable polymerase activities which differed in their cation requirement and substrate specificity were recovered with the virus-associated ribosomes. These polymerase activities synthesized homopolymers of limited chain length: in the presence of 10 mM Mg2%, polyuridylic acid was made, whereas in the presence of 1 mM Mn2%, polyadenylic acid was made. The addition of complementary RNA synthesized with the viral transcriptase in vitro to the reaction mixture containing the polyadenylic acid polymerase activity resulted in the terminal addition of polyadenylic acid to the complementary RNA. The possible function of the ribosome-associated polymerase activities in the replication of the virus is discussed.

Animals↗

PubMed2002

Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication.

Virions of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses contain conical cores consisting of a protein shell composed of the viral capsid protein (CA) surrounding an internal viral ribonucleoprotein complex. Although genetic studies have implicated CA in both early and late stages of the virus replication cycle, the mechanism of core disassembly following penetration of target cells remains undefined. Using quantitative assays for analyzing HIV-1 core stability in vitro, we identified point mutations in CA that either reduce or increase the stability of the HIV-1 core without impairing conical core formation in virions. Alterations in core stability resulted in severely attenuated HIV-1 replication and impaired reverse transcription in target cells with only minimal effects on viral DNA synthesis in permeabilized virions in vitro. We conclude that formation of a viral core of optimal stability is a prerequisite for efficient HIV-1 infection and suggest that disassembly of the HIV-1 core is a regulated step in infection that may be an attractive target for pharmacologic intervention.

Capsid↗

PubMed1994

Protection of retroviral DNA from autointegration: involvement of a cellular factor.

An essential step in the retrovirus life cycle is integration of a DNA copy of the viral genome into a host chromosome. After reverse transcription, there can be a delay of many hours before the viral DNA is integrated. It is important for the retrovirus to ensure that the viral DNA does not integrate into itself during this period; such autointegration is a suicidal process that would result in destruction of the viral genome. Understanding of the mechanism that blocks autointegration of the viral DNA may lead to insights into how to inhibit viral replication by inducing the viral DNA to autointegrate. Evidence is presented in this report that viral nucleoprotein complexes isolated from cells infected with Moloney murine leukemia virus exhibit a barrier to autointegration. The barrier can be disrupted by high salt treatment and, subsequently, restored by addition of factors provided by a host cell extract. Our data indicate an involvement of host machinery in protecting retroviral DNA from autointegration.

3T3 Cells↗

PubMed1999

Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro.

Primary human hepatocytes were used to develop a culture model for in vitro propagation of hepatitis C virus (HCV). Production of positive- strand full-length viral RNA in cells and culture supernatants was monitored by PCR methods targeting three regions of the viral genome: the 5' NCR, the 3' X-tail and the envelope glycoprotein E2. De novo synthesis of negative-strand RNA was also demonstrated. Evidence for a gradual increase in viral components over a 3 month period was obtained by two quantitative assays: one for evaluation of genomic titre (quantitative PCR) and one for detection of the core antigen. Production of infectious viral particles was indicated by passage of infection to naive hepatocyte cultures. Reproducibility of the experiments was assessed using cultures from three liver donors and eleven sera. Neither the genotype, nor the genomic titre, nor the anti-HCV antibody content, were reliable predictive factors of serum infectivity, while the liver donor appeared to play a role in the establishment of HCV replication. Quasispecies present in hepatocyte cultures established from three different liver donors were analysed by sequencing hypervariable region 1 of the E2 protein. In all three cases, the complexity of viral quasispecies decreased after in vitro infection, but the major sequences recovered were different. These data strongly suggest that human primary hepatocytes are a valuable model for study of persistent and complete HCV replication in vitro and for identification of the factors (viral and/or cellular) associated with successful infection.

Adult↗

PubMed1998

A role for human immunodeficiency virus type 1 Vpr during infection of peripheral blood mononuclear cells.

Studies analysing human immunodeficiency virus type 1 replication in primary cells have demonstrated that Vpr, although dispensable, plays a role along with the matrix (MA) protein in allowing nuclear localization of viral preintegration complexes in non-dividing monocyte-derived macrophages (MDMs). In the current study, experimental infection conditions to analyse the role of Vpr, independently of MA, during infection of PHA/IL-2-stimulated peripheral blood mononuclear cells (PBMC) were designed. It was shown that the absence of Vpr results in a subtle effect on virus production in long-term infection. PCR analysis of the steps of virus retrotranscription during a single cycle of replication in stimulated PBMC revealed that the absence of Vpr alone correlates with an impairment in the nuclear localization of viral DNA. Our data indicate that Vpr is involved in the virus life-cycle during infection of dividing PBMC, presumably as it is during infection of MDMs.

Cells, Cultured↗

PubMed1990

Solubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses.

Enveloped viruses, of which the orthomyxo- and paramyxoviruses are members, are known to be uncoated by nonionic detergents in a salt concentration-dependent manner. In this study we have shown that detergent uncoating of myxoviruses depends not only on salt concentration but also on pH. Treatment of orthomyxoviruses with Nonidet-P40 or Triton N-101 at low salt concentrations results in solubilization of surface virion glycopolypeptides in alkaline and neutral pH (9.0-6.5), but in acidic pH (6.0-5.0) the viral matrix protein M1 is also removed, and the viral ribonucleoprotein complex is released. Conversely, the paramyxovirus matrix protein M is more completely solubilized in alkaline pH (pH 9.0) than in neutral and acidic pH 7.4-5.0. The described pH-dependent differences are discussed in terms of orthomyxo- and paramyxovirus uncoating in target cells.

Animals↗

PubMed1969

Nonpermissive infections of mammalian cells: synthesis of inluenza virus genome in HeLa cells.

The RNA synthesis of a single-stranded, multisegmented viral genome has been investigated in nonpermissive host cells. In HeLa cells, both single- and double-stranded influenza viral RNA were made and accumulated, and synthesis was completed by 15 hours. The evidence indicated that all pieces of the genome were synthesized. At least as much viral RNA was synthesized in HeLa cells as in permissive chick embryo fibroblasts cells. Explanations for this nonpermissive infection based on reduced synthesis of viral RNA or rapid destruction of newly synthesized RNA, uncontrolled synthesis of RNA, or partial synthesis of the genome appear to have been ruled out by this investigation. The persistently high amounts of double-stranded RNA, combined with the reported lack of dispersion of nucleocapsid protein from the nuclear region of the HeLa cell are consistent with a failure of assembly of the virion in this host. This nonpermissive infection of HeLa cells with influenza virus may offer a unique system for the accumulation of the intermediates involved in the assembly of a complex RNA viral genome.

Animals↗

PubMed1999

Somatic cell mutants resistant to retrovirus replication: intracellular blocks during the early stages of infection.

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

Animals↗

PubMed1998

Protease cleavage of reovirus capsid protein mu1/mu1C is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle.

Mammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein sigma3 and contain protein mu1/mu1C as endoprotease-generated fragments mu1delta/delta and phi. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein mu1 in both these steps. To determine whether the cleavage of mu1/mu1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked sigma3 yet retained mu1/mu1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of mu1/mu1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of mu1/mu1C to mu1delta/delta and phi during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of mu1/mu1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.

Animals↗

PubMed2004

Reverse genetics of mononegavirales.

"Reverse genetics" or de novo synthesis of nonsegmented negative-sense RNA viruses (Mononegavirales) from cloned cDNA has become a reliable technique to study this group of medically important viruses. Since the first generation of a negative-sense RNA virus entirely from cDNA in 1994, reverse genetics systems have been established for members of most genera of the Rhabdo-, Paramyxo-, and Filoviridae families. These systems are based on intracellular transcription of viral full-length RNAs and simultaneous expression of viral proteins required to form the typical viral ribonucleoprotein complex (RNP). These systems are powerful tools to study all aspects of the virus life cycle as well as the roles of virus proteins in virus-host interplay and pathogenicity. In addition, recombinant viruses can be designed to have specific properties that make them attractive as biotechnological tools and live vaccines.

Animals↗

PubMed2006

The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication.

The results presented here demonstrate that the MHV and SARS-CoV nsp2 proteins are not required for the production of infectious virus, for polyprotein expression or processing, or for viral replication complex formation in cell culture. The nsp2 protein domain resides in a region of the coronavirus replicase that is relatively nonconserved across coronaviruses. In fact, the size and amino acid sequence variability of nsp2 across the different coronaviruses has led some investigators to speculate that the nsp2 protein, along with the nsp1 and nsp3 proteins, may play host- and/or cell-specific roles in the virus life cycle. While this may be the case, it should be noted that nsp2, in some form, exists in all coronaviruses studied to date and likely plays a pivotal role in the viral life cycle. A previous study from our laboratory identified a coronavirus replicase protein that plays an important role in viral pathogenesis. Such may prove to be the case for nsp2, as well. Alternatively, beacuse nsp2 exists as a detectable precursor protein nsp2-3 prior to processing of nsp2 and nsp3 into mature proteins, nsp2 may play a critical adaptor/regulatory role for nsp3 function. Importantly, the viruses produced in this study provide a system by which the role of the nsp2 protein in viral infection can be characterized.

Animals↗

PubMed1992

Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro.

We have investigated the mechanisms by which alleles at the mouse Fv-1 locus restrict replication of murine leukemia viruses. Inhibition of productive infection is closely paralleled by reduced accumulation of integrated proviral DNA as well as by reduced levels of linear viral DNA in a cytoplasmic fraction. Nevertheless, viral DNA is present at nearly normal levels in a nuclear fraction, and total amounts of viral DNA are only mildly affected in restrictive infections, suggesting a block in integration to account for reduced levels of proviral DNA. However, integrase (IN)-dependent trimming of 3' ends of viral DNA occurs normally in vivo during restrictive infections, demonstrating that not all IN-mediated events are prevented in vivo. Furthermore, viral integration complexes present in nuclear extracts of infected restrictive cells are fully competent to integrate their DNA into a heterologous target in vitro. Thus, the Fv-1-dependent activity that restricts integration in vivo may be lost in vitro; alternatively, Fv-1 restriction may prevent a step required for integration in vivo that is bypassed in vitro.

3T3 Cells↗

PubMed1988

Bacterial expression and characterization of nine polypeptides encoded by segments of the envelope gene of human immunodeficiency virus.

Nine envelope (Env) polypeptides, encoding different regions of HIV gp120 and gp41 Env proteins, and accounting for approx. 96% of the entire Env precursor glycoprotein complex (gp160) were expressed in Escherichia coli at levels ranging from approx. 2 to 20% of total cellular protein. The recombinant polypeptides were produced either as hybrid products fused to the cII gene fragment of the lambda vector or in an unfused form without interfering cII products. Partially purified protein fractions of each polypeptide were characterized serologically by Western-blot analysis against a panel of well characterized human immunodeficiency virus (HIV)-positive human reference sera. Most of the Env polypeptides were highly immunoreactive with anti-gp120/gp41 antibodies present in the sera of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases, but the patterns of reactivity were different. These results demonstrate that some of the antigenic determinants residing on the viral gp160 complex are retained on the surfaces of the recombinant Env polypeptides, and suggest that these sites are differentially immunogenic. These results are therefore interpreted in the context of an ongoing process towards using bacterially expressed HIV Env polypeptides to help define biological and structural epitopes to aid in the development of more sensitive diagnostic and therapeutic reagents in the fight against AIDS.

Cloning, Molecular↗

PubMed1989

RNA-binding properties of influenza A virus matrix protein M1.

The matrix protein (M1) of influenza A virus, which has a critical role in viral assembly and can inhibit the viral transcriptase complex, has the ability to bind RNA. The RNA-binding property of M1 is specific for single-stranded RNA, like that of influenza nucleoprotein (NP) and shows similar sensitivity to pH and to salt concentration. M1:RNA complexes are stable, once formed, to competition from excess single-stranded RNA. The possible location of the RNA-binding regions in the M1 protein is discussed.

Animals↗

PubMed1994

Involvement of the cyclic AMP-responsive element binding protein in bovine leukemia virus expression in vivo.

The TAR element (Tax-responsive element; also called TxRE) is a major determinant of the regulation of bovine leukemia virus (BLV) expression. In order to gain insight into the mechanisms of viral expression, complexes formed between proteins and the TAR enhancer DNA were analyzed by gel retardation assays. We report here that nuclear lysates from ex vivo-isolated B lymphocytes contain proteins that specifically bind to TAR. An antibody directed toward the cyclic AMP-responsive element binding (CREB) protein supershifted a complex (C1) present only in BLV-infected B lymphocytes. The CREB protein thus appears to be a major transcription factor involved in BLV expression in vivo.

Animals↗