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PubMed2006

Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex.

The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

Animals↗

PubMed1987

Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant-infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope.

Animals↗

PubMed2001

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.

Amino Acid Sequence↗

PubMed1999

A novel nuclear export activity in HIV-1 matrix protein required for viral replication.

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.

Biological Transport↗

PubMed1983

The leader RNA of vesicular stomatitis virus is bound by a cellular protein reactive with anti-La lupus antibodies.

The leader RNA transcript of vesicular stomatitis virus (VSV) has been immunoprecipitated from infected BHK cell extracts by anti-La specific sera from patients with systemic lupus erythematosus (SLE). This association was specific as lupus anti-sera with other specificities failed to precipitate leader RNA. The amount of leader RNA associated with the La antigen peaked 4 hr post infection and then declined. Leader RNA complexed with viral nucleocapsid proteins increased at a slower rate but eventually predominated 6 hr post infection. By 16 hr all of the leader RNA was associated with nucleocapsid proteins. Although a significant portion of the leader RNA was present in isolated nuclei 4 hr post infection, all of the leader RNA outside the nucleus was bound to La protein. Leader RNA is the first non-RNA polymerase III product found to associate with the La protein. The proposed function of the leader-La complex in VSV transcription and replication and in viral cytopathology is discussed.

Amino Acid Sequence↗

PubMed2004

A stochastic model of cytotoxic T cell responses.

We have constructed a stochastic stage-structured model of the cytotoxic T lymphocyte (CTL) response to antigen and the maintenance of immunological memory. The model follows the dynamics of a viral infection and the stimulation, proliferation, and differentiation of naïve CD8(+) T cells into effector CTL, which can eliminate virally infected cells. The model is capable of following the dynamics of multiple T cell clones, each with a T cell receptor represented by a digit string. MHC-viral peptide complexes are also represented by strings and a string match rule is used to compute the affinity of a T cell receptor for a viral epitope. The avidities of interactions are also computed by taking into consideration the density of MHC-viral peptides on the surface of an infected cell. Lastly, the model allows the probability of T cell stimulation to depend on avidity but also incorporates the notion of an antigen-independent programmed proliferative response. We compare the model to experimental data on the cytotoxic T cell response to lymphocytic choriomeningitis virus infections.

Antigens, Viral↗

PubMed1996

Reexamination of the Sendai virus P protein domains required for RNA synthesis: a possible supplemental role for the P protein.

The Sendai virus P protein plays a central role in viral genome amplification and expression, forming complexes with the viral L protein to generate the polymerase (P-L) and unassembled N (P-N(o)). This latter complex prevents N from self-assembling illegitimately, i.e., independently of the concurrent assembly of a nascent viral genome, and is thought to represent the functional form of N in nucleocapsid assembly. Based upon earlier functional studies using an in vitro transcription/ replication system in which the P, L, and N proteins were coexpressed, we identified two regions of the P protein required for RNA synthesis, namely, the C-terminal 40% of the protein, and a second, apparently redundant domain near the N-terminus (either amino acids 1-77 or 78-145). The lack of sequence conservation in this second region, apart from overall negative charge, was reminiscent of the acidic activation domains of cellular transcription factors. However, we recently mapped a chaperone domain at the N-terminal of P (aa 33-41), which is required for stable complex formation with unassembled N(o) and, thus, for assembly and genome replication. In this present study we show that coexpression of N protein with P deletion mutants lacking this region (e.g., P delta 1-324) results in the sequestration of the mutant P by the illegitimately assembled form of N. As a consequence, P protein is unavailable for RNA synthesis from bona fide templates. We also find that in the absence of coexpressed N protein, the entire N-terminal 60% of the P protein is not required for mRNA synthesis. During these studies, a supplemental role for the P protein in viral RNA synthesis, independent of stable complex formation with L, was observed. This function involves, at least in part, the binding of additional copies of P to the N:RNA template.

Binding Sites↗

PubMed1996

Vpx association with mature core structures of HIV-2.

Vpx is a virion-incorporated protein encoded by HIV-2 and certain strains of SIV which is necessary for efficient infection of lymphocytes and macrophages. To determine the location of Vpx within the HIV-2 particle, core structures were obtained from mature HIV-2 virions. Analysis of purified cores reveal the presence of monomeric Vpx as well as a nondenaturable and nonreducible higher order Vpx complex. Characterization of the core-associated Vpx complex suggests it to be a Vpx homodimer, indicative of high concentrations of Vpx. The incorporation and concentration of Vpx in HIV-2 cores support the hypothesis that it functions within the viral preintegration complex.

Animals↗

PubMed2003

Analysis of the full-length integrase-DNA complex by a modified approach for DNA docking.

A model of the full-length HIV-1 integrase dimer was constructed assembling the experimentally determined structures of the single domains. Subsequently, the three-domain protein-viral DNA complex was generated for the first time through an automated docking algorithm, obtained modifying the ESCHER program, a well-known method for protein-protein docking. A detailed study of the contacts established with DNA by the enzyme revealed that the predicted model reproduced the results of mutagenesis and cross-linking experiments, confirming the validity of our docking approach in predicting the base specificity in the DNA-protein interaction.

Base Sequence↗

PubMed2006

Effects of brefeldin A on the localization of Tobamovirus movement protein and cell-to-cell movement of the virus.

It has been demonstrated that the subcellular location of Tobamovirus movement protein (MP) which was fused with green fluorescent protein (MP:GFP) changed during the infection process. However, the intracellular route through which MP is transported and its biological meaning are still obscure. Treatment with brefeldin A (BFA), which disrupts ER-to-Golgi transport, inhibited the formation of irregularly shaped and filamentous structures of MP. In this condition, MP was still targeted to plasmodesmata in leaf cells. Furthermore, the viral cell-to-cell movement was not inhibited by BFA treatment. These data indicated that the targeting of viral replication complexes (VRCs) to plasmodesmata is mediated by a BFA-insensitive pathway and that the ER-to-Golgi transport pathway is not involved in viral intercellular movement.

Antiviral Agents↗

PubMed1997

HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import.

Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.

Amino Acid Sequence↗

PubMed1993

Heterocomplexes of tick-borne encephalitis structural and non-structural proteins.

The existence of the complex of structural and non-structural proteins in the tick-borne encephalitis (TBE) virus is shown. The complex was isolated from virus-containing cultural medium by immunoaffinity chromatography on monoclonal antibodies (MAbs). By enzyme immunoassay and immunoblotting with the use of appropriate MAbs it was demonstrated that this complex consists of structural (protein E), and non-structural (NS1) glycoproteins. Also, the trimer E-NS1-NS3 can be isolated. It is proposed that this trimer is the viral replicative complex.

Antibodies, Monoclonal↗

PubMed1983

Interferon pretreatment inhibits simian virus 40 infections by blocking the onset of early transcription.

CV-1 cells treated with human leukocyte interferon before infection with simian virus 40 (SV40) are protected from virus-induced cytopathic effects. Accumulation of viral mRNA is inhibited markedly. If T antigen is present in interferon-treated cells before infection, viral DNA replication is inhibited only slightly. These results show that many SV40 virions reach the nucleus in a form which can be replicated but not transcribed. Accumulation of SV40 mRNA is not inhibited in cells treated with interferon after infection. Thus the block mediated by interferon is specific for the onset of transcription. Infections with SV40 DNA rather than virions are insensitive to treatment with interferon, showing that the inhibition involves an interaction between viral nucleoprotein complexes and cellular factors.

Animals↗

PubMed1977

The sv40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription.

A small fraction of the SV40 chromatin isolated from infected monkey cell cultures by the Triton method contains active RNA polymerase which had initiated transcription in vivo. This viral transcription complex (VTC) was utilized to answer the question of whether proteins dissociate from chromatin during transcription in vitro. 3H-RNA was synthesized by the VTC under conditions such that over half the label was in transcripts which were longer than half the length of the SV40 genome. Virtually all of the 3H-RNA remained associated with the SV40 chromatin, causing an increase in sedimentation rate from 55S to 78S. The density of the VTC-3H-RNA complex indicated that less than 5% of the original protein dissociated from the SV40 DNA which served as a template for transcription. We conclude that SV40 chromatin can be transcribed while the proteins remain associated with the DNA.

Cell Line↗

PubMedDate not supplied

The host environment drives HIV-1 fitness.

Viral fitness is defined by the ability of an individual genotype to produce infectious progeny in a specific environment. For HIV the environment is never constant but rather fluctuates in time and space. For instance, environmental factors that determine viral fitness during transmission from host to host are different to the pressures from either cytotoxic T-lymphocytes (CTLs) or antiviral drugs. Consequently, viral fitness is highly dependent on the environment and the accurate determination of this value therefore depends strongly on the chosen environmental setting. This review describes how the host environment imposes selective pressures on the virus that shape its genotype and fitness. The most important environments that the virus encounters throughout its life cycle and during natural infection are discussed. In order of appearance, CTLs are discussed, followed by neutralising antibodies and antiretroviral drug treatment. It then goes on to describe receptor molecules that mediate viral entry and intracellular restriction factors, which represent selective pressures that are present directly from the start of a natural infection. It concludes by discussing the complexity of viral fitness and how an accurate measure of viral fitness eventually may, for example, contribute to the improvement of antiretroviral therapy or help in the formulation of an optimal vaccination strategy.

Adaptation, Biological↗

PubMed1982

Attenuation in the control of SV40 gene expression.

Nuclei and viral transcriptional complexes were prepared from cells infected with simian virus 40 and incubated in vitro in the presence of alpha- 32P-UTP. The in vitro elongated viral RNA appeared with a peak of 5S in sucrose gradients and hybridized preferentially to a promoter-proximal region of SV40 DNA. Treatment of infected cells with proflavine led to transcription of elongated RNA, while treatment of cells with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a drug known to enhance premature termination, augmented accumulation of the promoter-proximal RNA. The in vitro elongated RNA produced a major band of 93-95 nucleotides in length in acrylamide gel. This RNA was found to map between the major initiation site at nucleotide 243 and nucleotides 335-337. The significance of these observations with respect to the transcription termination signal and the control of SV40 gene expression is discussed.

Animals↗

PubMed2006

The NPro product of bovine viral diarrhea virus inhibits DNA binding by interferon regulatory factor 3 and targets it for proteasomal degradation.

Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of interferon regulatory factor 3 (IRF-3), a transcription factor essential for interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I interferon induction or the targeting of IRF-3 for degradation.

Animals↗

PubMed1981

DNA synthesis and template activity in a mutant of polyoma virus with altered expression of late viral proteins.

The 3049 strain of polyoma virus overproduces late RNA and proteins. The synthesis and accumulation of virus DNA was measured in cells infected with 3049 and a wild-type virus, lpS, to assess the possible role of gene dosage in this phenomenon. The rate of incorporation of [3H]thymidine into the DNA of 3049 and lpS virus was shown to be identical. The number of genome copies per cell, determined by DNA-DNA reassociation kinetics, was found to be similar in either whole cell or Hirt extracts of cells infected with the two viruses. Viral transcription complexes were isolated by Sarkosyl extraction, and the specific transcriptional activity was measured by incorporation of [3H]UTP. The incorporation of [3H]UTP and the DNA content of transcription complexes were indistinguishable. These results suggest that overproduction of late RNA (and protein) by the 3049 virus is not due to either an increased number of viral genomes per cell or an increased fraction of genomes involved in transcription. These and other data (unpublished) support the conclusion that the altered phenotype is due to a posttranscriptional mechanism.

Animals↗