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At least 991 records · Page 55Linked to original sources
PubMed1991

Expression strategy of the potato virus X triple gene block.

The mode of expression of the overlapping genes of the triple block positioned internally in potato virus X (PVX) RNA was examined. The results of In vitro translation of synthetic RNA transcripts and natural PVX-specific methylmercuric hydroxide-denatured dsRNAs suggest that the 25K protein is expressed as a single translation product of the 2.1 kb subgenomic (sg) RNA and that both the 12K and 8K proteins are expressed from the same 1.4 kb sgRNA.

Base Sequence↗
PubMed1981

Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene.

The RNA of a unique long defective interfering particle (DI-LT2) derived from the heat-resistant strain of vesicular stomatitis virus (VSV) contains 70 nucleotides at its 3' end that are complementary to the 5' end of the VSV RNA. Following this region of terminal complementarity, there is a precise copy of the 3' end of the nondefective VSV RNA. The sequence homology between the DI-LT2 RNA and the 3' end of VSV RNA extends for at least 60 bases and probably for most of the length of the DI-LT2 RNA. The DI-LT2 particle is capable of transcription in vitro but produces only a short RNA [defective interfering (DI) particle product], which is encoded by the extreme 3' terminus of the DI RNA. Neither leader RNA nor capped VSV mRNAs are synthesized by DI-LT2, although competent templates for these are present. These data suggest that the 3'-terminal initiation is a prerequisite of the production of competent transcripts and that the sequence coding for leader RNA is not, by itself, sufficient for initiation. We propose a model for the origin of this DI particle, involving specific termination and resumption of replication, which is similar to that described previously for another class of DI particle RNAs.

Animals↗
PubMed1990

The phosphorylation of the integral membrane (M1) protein of influenza virus.

The phosphorylation of the internal and integral membrane (M1) protein of influenza virus was studied. Four points can be made based on the data: (1) The M1 contains at least two moles of phosphate per mole of M1. (2) Phosphorylation of M1 is conserved between influenza A, B and C viruses. Other characteristics of the M1 are also conserved, such as solubility in organic solvent, heterogeneity and ability to partition into lipid vesicles. (3) M1 is phosphorylated in cells infected with a vaccinia recombinant (vP273) containing only the gene of M1, either as a result of a vaccinia virus associated kinase or a cellular one. (4) The phosphate is located within or in close proximity to the major stretch of neutral and hydrophobic amino acids found in M1, as determined by analyzing cyanogen bromide fragments.

Amino Acid Sequence↗
PubMed1999

Quantitative effects of valacyclovir on the replication of cytomegalovirus (CMV) in persons with advanced human immunodeficiency virus disease: baseline CMV load dictates time to disease and survival. The AIDS Clinical Trials Group 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group.

Virus load is a major risk factor for disease in many human viral infections, especially human immunodeficiency virus (HIV) disease. The effect of cytomegalovirus (CMV) load on disease progression and the influence of antiviral chemotherapy on surrogate markers of replication was investigated in 310 patients with advanced HIV disease in a randomized controlled trial that compared the effects of valacyclovir with those of acyclovir. Sequential blood and urine samples were analyzed by polymerase chain reaction (PCR), for human CMV (HCMV) DNA. In multivariate analyses, elevated virus load in both blood and urine at baseline was associated with increased risk of HCMV disease (relative hazard, 1.49 and 1.44 per log increase, respectively). Elevated virus load in blood at baseline was also associated with a significantly shorter survival time (log rank, P=. 0001). In time-updated analyses, valacyclovir significantly suppressed the virus load in subjects who were PCR positive at baseline (in blood or urine), when compared with the combined acyclovir arms.

AIDS-Related Opportunistic Infections↗
PubMed1984

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.

Amino Acid Sequence↗
PubMed2005

Enhancement of internal ribosome entry site-mediated translation and replication of hepatitis C virus by PD98059.

Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IRES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication.

Base Sequence↗
PubMed1998

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence.

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

Amino Acid Sequence↗
PubMed2004

Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control.

A method for quantitation of hepatitis C virus (HCV) RNA was developed based on competitive reverse transcription polymerase chain reaction (RT-PCR) using in vitro transcribed mutated thio-RNA as a competitor template. The thio-RNA is more resistant to RNAse and is stable over a year. This assay was compared with the commercially available Roche Amplicor HCV Monitor assay V 2.0 and real time PCR using SYBR green 1 dye method. A total of 18 pre-therapy serum samples from chronic hepatitis C cases were tested in parallel by the three assays. All samples could be quantitated using the in-house competitive RT-PCR and real time PCR and there was a significant correlation in the virus titer (P<0.05). However, 8 (44%) samples could not be quantified by Amplicor HCV Monitor assay, which has a lower detection range (10(2) to 10(5.5) copies/ml). The in-house method of competitive RT-PCR showed a detection range of 10(3) to 10(10) copies/ml. In the patients the mean viral titer was found to be (9.66+/-9.3)x10(6) copies/ml. Ten (55%) of the samples, assessed by the Amplicor HCV Monitor assay showed a mean viral titre of (1.13+/-0.75)x10(6) copies/ml, which was lower than the other two tests. The competitive PCR method and real time PCR could amplify all prevalent genotypes. This in-house quantitative competitive RT-PCR method is simple, cheap, reproducible and useful for estimation of HCV RNA load.

Genotype↗
PubMed1995

Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1.

The internal structural proteins of retroviruses are proteolytically processed from the Gag polyprotein, which alone is able to assemble into virus-like particles when expressed in cells. All Gag proteins contain domains corresponding to the three structural proteins MA, CA, and NC. We have expressed the CA and NC domains together as a unit in Escherichia coli, both for Rous sarcoma virus (RSV) and for human immunodeficiency virus type 1 (HIV-1). We also expressed a similar HIV-1 protein carrying the C-terminal p6 domain. RSV CA-NC, HIV-1 CA-NC, and HIV-1 CA-NC-p6 were purified in native form by classic methods. After adjustment of the pH and salt concentration, each of these proteins was found to assemble at a low level of efficiency into structures that resembled circular sheets and roughly spherical particles. The presence of RNA dramatically increased the efficiency of assembly, and in this case all three proteins formed hollow, cylindrical particles whose lengths were determined by the size of the RNA. The optimal pH at which assembly occurred was 5.5 for the RSV protein and 8.0 for the HIV-1 proteins. The treatment of the RSV CA-NC cylindrical particles with nonionic detergent, with ribonuclease, or with viral protease caused disassembly. These results suggest that RNA plays an important structural role in the virion and that it may initiate and organize the assembly process. The in vitro system described should facilitate the dissection of assembly pathways in retroviruses.

Amino Acid Sequence↗
PubMed2002

Out-of-frame versus in-frame core internal deletion variants of human and woodchuck hepatitis B viruses.

Human hepatitis B virus (HBV) variants containing in-frame core internal deletion (CID) have been demonstrated to contain all the functional features of defective interfering (DI) particles (Yuan, T. T.-T., M.-H. Lin, D. S. Chen, and C. Shih, 1998, J. Virol. 72, 578-584). Here, we report that out-of-frame HBV CID variants exhibit defective interfering property similar to in-frame CID variants characterized previously. This result raises the possibility that it may be the deleted pregenomic RNA product, rather than the deleted core protein product, that is responsible for interference. Furthermore, a genomic deletion elsewhere does not cause interference since preS2 deletion variants exhibit no influence on wild-type HBV replication. Consistent with the natural occurrence of HBV CID variants, we recently identified CID variants of woodchuck hepatitis virus (WHV) in natural infection. However, unlike HBV CID variants, functional characterization of WHV CID variants using a human hepatoma cell line has not revealed any interference in tissue culture. In summary, defective interference is a general phenomenon for both in-frame and out-of-frame HBV CID variants.

Animals↗
PubMed2004

Binding, internalization, and membrane incorporation of human immunodeficiency virus-1 at the blood-brain barrier is differentially regulated.

Human immunodeficiency virus (HIV)-1 within the CNS induces neuro-acquired immunodeficiency syndrome and acts as a reservoir for reinfection of peripheral tissues. HIV-1 crosses the blood-brain barrier (BBB) within infected immune cells and as cell-free virus by a CD4-independent mechanism. Which proteins control free virus transport across the BBB are unknown, but work with wheatgerm agglutinin (WGA) and heparin suggests that heparan sulfate proteoglycans, sialic acid, and N-acetyl-beta-D-glucosaminyl acid bind HIV-1. Here, we found that an HIV-1 T-tropic virus was taken up by mouse brain endothelial cells in vitro and crossed the BBB in vivo and could be effluxed as intact virus. Uptake was stimulated by WGA and protamine sulfate (PS) and inhibited by heparin. BBB uptake of virus involved four distinguishable binding sites: i) reversible cell surface binding involving gp120 and sensitive to PS/heparin but insensitive to WGA; internalization with a ii) WGA-sensitive site binding gp120 and iii) a PS/heparin-sensitive site not involving gp120; iv) membrane incorporation not affected by WGA, heparin, or PS. In conclusion, binding, internalization, and membrane incorporation are separately regulated steps likely determining whether HIV-1 is incorporated into brain endothelial cells, transported across them, or returned to the circulation.

Animals↗
PubMed2007

WHO Rapid Advice Guidelines for pharmacological management of sporadic human infection with avian influenza A (H5N1) virus.

Recent spread of avian influenza A (H5N1) virus to poultry and wild birds has increased the threat of human infections with H5N1 virus worldwide. Despite international agreement to stockpile antivirals, evidence-based guidelines for their use do not exist. WHO assembled an international multidisciplinary panel to develop rapid advice for the pharmacological management of human H5N1 virus infection in the current pandemic alert period. A transparent methodological guideline process on the basis of the Grading Recommendations, Assessment, Development and Evaluation (GRADE) approach was used to develop evidence-based guidelines. Our development of specific recommendations for treatment and chemoprophylaxis of sporadic H5N1 infection resulted from the benefits, harms, burden, and cost of interventions in several patient and exposure groups. Overall, the quality of the underlying evidence for all recommendations was rated as very low because it was based on small case series of H5N1 patients, on extrapolation from preclinical studies, and high quality studies of seasonal influenza. A strong recommendation to treat H5N1 patients with oseltamivir was made in part because of the severity of the disease. Similarly, strong recommendations were made to use neuraminidase inhibitors as chemoprophylaxis in high-risk exposure populations. Emergence of other novel influenza A viral subtypes with pandemic potential, or changes in the pathogenicity of H5N1 virus strains, will require an update of these guidelines and WHO will be monitoring this closely.

Animals↗
PubMed2005

Hepatitis C virus core protein acts as a trans-modulating factor on internal translation initiation of the viral RNA.

Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5' end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression.

Gene Expression Regulation, Viral↗
PubMed1987

Human epithelial cell expression of an Epstein-Barr virus receptor.

Cultured human epithelium bound and internalized radiolabelled Epstein-Barr virus (EBV) within 1 h of exposure. A similar percentage of cultured cells also were reactive with monoclonal antibodies to the EBV/C3d receptor of B lymphocytes. In cross-sections of fresh frozen, stratified epithelium, receptor expression seemed limited to the less differentiated subpopulation of cells. These findings support the notion of direct infection of epithelial cells by EBV and suggest a viral life cycle in epithelium dependent on the stage of cell differentiation.

Cell Line↗
PubMed1978

An infectivity assay for the bovine leukemia virus based on the induction of the major internal virion antigen in susceptible cell cultures.

This report described the development of a 7-day infectivity assay for the bovine leukemia virus (BLV) which is based on the induction of the major internal virion antigen (p25) in susceptible indicator monolayer cell cultures. In this assay the antigen is detected in the indicator cells by the immunoperoxidase antibody technique using a monospecific anti-BLV serum. The immunoperoxidase infectivity assay (IPIA) is specific, quantitative, reproducible and more sensitive than the previously developed syncytia induction assay. The IPIA can be applied for the detection of BLV-infected cells and provides a reliable method for the direct diagnosis of BLV infection in cattle.

Antigens, Viral↗