SEARCH · PubMed Health

Results for “viral complexity”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources

PubMed2004

The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein.

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.

Antiviral Agents↗

PubMed2004

Role of Nup98 in nuclear entry of human immunodeficiency virus type 1 cDNA.

Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses, can infect non-dividing cells. The lentiviruses are most likely to have evolved a nuclear import strategy to import HIV-1 cDNA and viral protein complex through the nuclear pore complex (NPC) formed by nucleoporin proteins (Nup). In this study, we found that synthesis of integrated and 2LTR but not full-length form of HIV-1 cDNA was clearly impaired in culture via transduction of vesicular stomatitis virus matrix protein (VSV M), an inhibitor protein, through binding to the phenylalanine-glycine (FG) repeat region of Nup98. The impairment of synthesis of integrated and 2LTR DNA with VSV M was restored by ectopic overexpression of Nup98. A series of experiments using Nup98-depleted NPC by the small interfering RNA (siRNA) technique showed specific impairment of NPC structure and some functions, including nuclear import of HIV-1 cDNA. Our results suggest that Nup98 on the NPC specifically participates in the nuclear entry of HIV-1 cDNA following HIV-1 entry.

Active Transport, Cell Nucleus↗

PubMed1998

Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.

After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex.

Cell Line↗

PubMed2006

Rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA.

The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA complex is the template for viral transcription and replication. The viral phosphoprotein (P) has two functions during the infection process: it binds through its carboxy-terminus to N in the N-RNA complex and at the same time with an amino-terminal domain to the polymerase and in this way fixes the polymerase to its template. The second function of P is to bind to newly produced N in the infected cell in order to prevent that N binds non-specifically and irreversibly to cellular RNA. In order to identify the part of the phosphoprotein that binds to N and keeps the latter soluble, we isolated the N-P complex, performed sequential protease digestions, and determined the identity of the remaining N and P peptides in the purified digested complex. Although the digestion steps removed short sequences of N, most of N remained intact and soluble, indicating that the overall structure was not affected. Most of P, including the carboxy-terminal N-RNA-binding domain, was removed during the first digestion step. N-terminal sequencing and mass spectrometry analysis identified a P peptide containing residues 4-40 that remained associated with N. Coexpression and coimmunoprecipitation experiments and yeast two-hybrid experiments showed that this peptide alone could bind to N in vivo.

Amino Acid Sequence↗

PubMed2000

Cell cycle-dependent stimulation of the HIV-1 promoter by Tat-associated CAK activator.

Activation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat-TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436-7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 --> G1 progression with the expression of dominant negative mutant Ras(Asn17) or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor.

Cell Cycle↗

PubMed2005

Wrapping things up about virus RNA replication.

All single-stranded 'positive-sense' RNA viruses that infect mammalian, insect or plant cells rearrange internal cellular membranes to provide an environment facilitating virus replication. A striking feature of these unique membrane structures is the induction of 70-100 nm vesicles (either free within the cytoplasm, associated with other induced vesicles or bound within a surrounding membrane) harbouring the viral replication complex (RC). Although similar in appearance, the cellular composition of these vesicles appears to vary for different viruses, implying different organelle origins for the intracellular sites of viral RNA replication. Genetic analysis has revealed that induction of these membrane structures can be attributed to a particular viral gene product, usually a non-structural protein. This review will highlight our current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA-membrane interactions observed between the virus families Flaviviridae and Picornaviridae.

Animals↗

PubMed2002

Infection of macaques with a molecular clone, SHIVSF33A2, provides evidence for tissue specific variants.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established model to study acquired immunodeficiency syndrome (AIDS) pathogenesis. Such a controlled system allows for detailed analysis of the molecular determinants of viral pathogenesis in addition to studying host-specific immune responses that modulate disease progression. Furthermore, the use of a pathogenic molecular clone affords the opportunity to study both viral evolution within a host and to examine the generation of tissue specific variants. In this report we describe viral diversification within tissues of two rhesus macaques infected intravenously with the CXCR4-specific molecular clone SHIVSF33A2. Heteroduplex tracking analysis (HTA) was used to determine the complexity of viral DNA within distinct lymphoid tissues. Not surprising, heterogeneity of the proviral quasispecies in tissues obtained during the acute infection was limited. However, tissues obtained at necropsy harbored a more diverse and often different population of env variants. As the inoculating virus is a molecular clone, the variants generated are likely due to the presence of tissue specific selective forces rather than a founder's effect.

Animals↗

PubMed1995

Phase I/II study of 3TC (lamivudine) in HIV-positive, asymptomatic or mild AIDS-related complex patients: sustained reduction in viral markers. The Lamivudine European HIV Working Group.

OBJECTIVE: To evaluate the efficacy of 3TC (lamivudine), a synthetic nucleoside analogue that inhibits HIV reverse transcriptase in vitro, as treatment for HIV-positive, asymptomatic or mild AIDS-related complex patients. DESIGN: Open-label, multinational and multicentre, non-comparative, escalating dose study. METHODS: Patients who meet the selection criteria (n = 104) were enrolled in three European countries. Ten to 15 patients were included at each of the six dose levels of 3TC (0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 20.0 mg/kg daily in two divided doses every 12 h). Virological parameters--immune-complex dissociation (ICD) assay for HIV p24 antigenaemia, plasma HIV RNA load, whole blood assay and cellular viraemia--were evaluated at weeks 0, 4, 12 and 24. RESULTS: Sustained reductions in HIV RNA load and in ICD p24 antigen levels were observed and maintained over the 12-week assessment period. Greater reductions were noted at higher doses but this trend did not reach statistical significance. In 38 patients, reductions of cell viraemia were significantly greater at 4 weeks for patients treated at higher doses of 3TC. CONCLUSION: These virological data show that 3TC is a potent inhibitor of HIV replication in HIV-positive, asymptomatic or mild ARC patients as assessed by ICD p24 antigenaemia, plasma HIV RNA load and cell viraemia.

AIDS-Related Complex↗

PubMed2002

PNA targeting the PBS and A-loop sequences of HIV-1 genome destabilizes packaged tRNA3(Lys) in the virions and inhibits HIV-1 replication.

During assembly of the HIV-1 virions, cellular tRNA(Lys)(3) is packaged into the virion particles and is utilized as a primer for the initiation of reverse transcription. The 3'-terminal 18 nucleotides of the cellular tRNA(Lys)(3) are complementary to nucleotides 183-201 of the viral RNA genome, referred to as the primer binding sequence (PBS). Additional sequences (A-Loop) upstream of the PBS are essential for tRNA primer selection. We report here that a PNA targeted to PBS and A-Loop sequence (PNA(PBS)) exhibits high specificity for its target sequence and prevents tRNA(Lys)(3) priming on the viral genome. We also demonstrate that PNA(PBS) is able to invade the duplex region of the tRNA(Lys)(3)-viral RNA complex and destabilize the priming process, thereby inhibiting the in vitro initiation of reverse transcription. The endogenously packaged tRNA(Lys)(3) bound to the PBS region of the viral RNA genome in the HIV-1 virion is efficiently competed out by PNA(PBS), resulting in near complete inhibition of initiation of endogenous reverse transcription. Examination of the effect of PNA(PBS) on HIV-1 production in CEM cells infected with pseudotyped HIV-1 virions carrying luciferase reporter exhibited dramatic reduction of HIV-1 replication by nearly 99%. Analysis of the mechanism of PNA(PBS)-mediated inhibition indicated that PNA(PBS) interferes at the step of reverse transcription. These findings suggest the antiviral efficacy of PNA(PBS) in blocking the process of HIV-1 replication.

Base Sequence↗

PubMed1987

Analysis of the genomic L RNA segment from lymphocytic choriomeningitis virus.

The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.

Amino Acid Sequence↗

PubMed1978

Transcription of host-substituted simian virus 40 DNA in whole cells and extracts.

Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40.

Base Sequence↗

PubMed1995

Double-stranded RNA bacteriophage phi 6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions.

Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.

Acid Anhydride Hydrolases↗

PubMed1996

Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5' splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5' splice site. Second, utilization of the weak M2 5' splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine-rich splicing enhancer sequence that is located in the 3' exon of M1 mRNA. We demonstrate that activation of the M2 5' splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.

Alternative Splicing↗

PubMed1999

The effect of viruses on the ability to present antigens via the major histocompatibility complex.

We describe viral pathogens that cause significant human disease by their ability to interfere with the expression of major histocompatibility complex class I and II molecules. Herpesviruses and papillomaviruses encode gene products that interfere with the class I pathway of antigen processing and/or peptide translocation. Adenoviruses encode unique gene products that interfere with transport of class I molecules. Influenza virus, measles virus, and HIV interfere with the class II pathway by either suppressing the production of class II molecules or impeding antigen trafficking. Cytomegalovirus interferes with both class I and class II pathways. Better understanding of these mechanisms may lead to further insight into the pathogenesis of viral infections and allow for improved treatments.

Antigen Presentation↗

PubMed2004

Mechanism of the interferon alpha response against hepatitis C virus replicons.

Interferon alpha (IFN-alpha) inhibits hepatitis C virus (HCV) replication in vivo and in cell cultures by one or several mechanisms that are not yet understood. We sought to identify the viral targets of the IFN-alpha-induced cellular antiviral program in Huh7 cells expressing HCV subgenomic replicons. Our results revealed a tight linkage between translation, assembly of replication complexes and viral RNA synthesis, and indicated that the stability of amplified plus strand RNA was reduced in the presence of the cytokine. Moreover, HCV internal ribosomal entry site (IRES)-directed translation was inhibited approximately 2-fold in IFN-treated cells. In contrast, the synthesis of viral RNA did not seem to be directly affected by the antiviral program induced by the cytokine. Our results were consistent with a model predicting that the IFN-alpha-induced antiviral program could inhibit multiple steps of the HCV replication cycle, leading to a reduction in viral protein synthesis and eventually inhibition of viral RNA amplification.

Hepacivirus↗

PubMed2005

Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: implication for modular evolution of RNA viruses.

The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and post-recombinational amplification of new recombinant viruses.

Base Sequence↗

PubMed1995

The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.

Replication of hepadnaviruses requires a persistent population of covalently closed circular (CCC) DNA molecules in the nucleus of the infected cell. It is widely accepted that the vital role of this molecule is to be the sole DNA template for the synthesis by RNA polymerase II of all viral transcripts throughout the infection process. Since the transcriptional activity of eukaryotic nuclear DNA is considered to be determined in part by its specific organization as chromatin, the nucleoprotein disposition of the hepadnavirus CCC DNA was investigated. These studies were undertaken on the duck hepatitis B virus (DHBV) CCC DNA present in the liver cell nuclei of DHBV-infected ducks. The organization and protein associations of the DHBV CCC DNA in situ were inferred from sedimentation, micrococcal nuclease digestion, and DNA superhelicity analyses. These three lines of investigation demonstrate that the DHBV CCC DNA is stably associated with proteins in the nuclei of infected liver cells. Moreover, they provide compelling evidence that the viral nucleoprotein complex is indeed a minichromosome composed of classical nucleosomes but in arrays that are atypical for chromatin. When the DHBV chromatin is digested with micrococcal nuclease, a ladder of viral DNA fragments that exhibits a 150-bp repeat is produced. This profile for the viral chromatin is obtained from the same nuclei in which the duck chromatin shows the standard 200-bp ladder. The superhelicity of the DHBV CCC DNA ranges from 0 to 20 negative supertwists per molecule, with all possible 21 topoisomers present in each DNA preparation. The 21 topoisomers of DHBV CCC DNA are inferred to derive from an identically diverse array of viral minichromosomes. In the DHBV minichromosomes composed of 20 nucleosomes, 96.7% of the viral DNA is calculated to be compacted into these chromatin subunits spaced on average by 5 bp of linker DNA; other minichromosomes contain fewer nucleosomes and proportionately more linker DNA. Two major subpopulations of DHBV minichromosomes are detected with comparable prevalence. The two groups correspond to minichromosomes which contain essentially a full or half complement of nucleosomes. The functional significance of this minichromosome diversity is unknown but is suggestive of transcriptional regulation of the viral DNA template.

Animals↗

PubMed1990

The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations.

The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB-viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non-contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein.

Adenovirus Early Proteins↗