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PubMed2003

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro.

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

Cerulenin↗
PubMed1983

Characteristics and regulation of interaction of avian retrovirus pp12 protein with viral RNA.

We investigated the interaction of the avian retrovirus pp12 protein with viral RNA to assess its possible role in virion assembly. Using chemical modification techniques, we found that reagents specific for lysine or arginine residues inactivated the RNA-binding capacity of the protein. The binding of pp12 to 60S viral RNA was also strongly affected by pH (pKapp of 5.5); the affinity for viral RNA decreased by as much as 40-fold after protonation of one or more titratable groups on the protein. When the protein was cleaved by cyanogen bromide, each of the two polypeptide products bound to RNA (with low affinity), but pH dependence was lost. Thus, an intact protein was required for this effect. Since histidine and phosphoserine residues have pKa values close to the pKapp of the pp12-RNA interaction, they were studied to determine whether they were involved in this process. Each of the two histidyl residues in pp12 had pKa values of 6.2, as determined by proton nuclear magnetic resonance titrations, values too high to account for the pKapp of binding. The involvement of phosphoserine residues, which have pKa values similar to the pKapp, was investigated by removal of phosphate from pp12. When phosphate groups were chemically or enzymatically removed from the avian myeloblastosis virus, Rous sarcoma virus (Pr-C), and PR-E 95C virus pp12 proteins, the Kapp for binding 60S viral RNA was reduced 100-fold at pH 7.5. Thus, it seems possible that phosphorylation of the pp12 protein could favor viral nucleocapsid formation by increasing its affinity for the viral RNA genome. Dephosphorylation could provide for its release from the viral RNA during reverse transcription after viral infection of cells.

Alpharetrovirus↗
PubMed2004

Purification of the cucumber necrosis virus replicase from yeast cells: role of coexpressed viral RNA in stimulation of replicase activity.

Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i). to initiate cRNA synthesis de novo on both plus- and minus-stranded templates, (ii). to generate replicase products that are shorter than full length by internal initiation, and (iii). to perform primer extension from the 3' end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.

Cucumis sativus↗
PubMed2003

Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates.

To identify the potential RNA inhibitors of HCV polymerase, we have isolated high-affinity RNA ligands specific to hepatitis C virus (HCV) NS5B protein from a combinatorial RNA library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) procedure. Thirty-seven selected ligands were classified into eight groups on the basis of their sequence homologies. Most (60%) of the ligands carry the conserved YGUAGR hexamer (Y = pyrimidine, R = purine) at the 5' end of the 40-nt randomized region, and 74% of the ligands end in (A/C)U at the 3'end. However, strong binding to NS5B required the whole RNA ligand including the flanking conserved nucleotides at both ends. The binding of the selected ligands to NS5B is highly specific and strong, as reflected in their low dissociation rate constants (k(d) approximately 10(-4) s(-1)). Analysis of secondary structure by computer program and RNase footprints of the two different aptamers from two most conserved groups revealed RNA structures containing three stem loops with internal bulges. NS5B bound these RNA at a region between the two stem loops from the 5' -end. Some of these RNA aptamers could serve as a template for the HCV polymerase, but some interfered with the activity of the viral enzyme. These RNA ligands will be useful for further characterization of NS5B-binding properties and, with further modifications, may have potential therapeutic value.

Base Sequence↗
PubMed2006

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

Plant Viruses↗
PubMed2004

Plasma RNA viral load is not associated with intrapatient quasispecies heterogeneity in HIV-1 infection.

The human immunodeficiency virus type 1 (HIV-1) viral set point has been associated with the rate of, disease progression and with the level of HIV-specific immune response. The analysis of the possible association between viral set point and quasispecies heterogeneity has important consequences in the understanding of HIV-1 in vivo evolution. In this study, we analyzed the association between intrapatient viral diversity and RNA viral load in 16 antiretroviral therapy-naive HIV-1-infected patients at a single time point, during the disease free period. Patients were separated into low and high viral load groups according to plasma RNA values. HIV-1 quasispecies complexity was assessed in the C2-V5 env region. The average intrapatient quasispecies heterogeneity in both groups was not significantly different (t-test, P > 0.05). However, while within the low viral load group both synonymous and non-synonymous mutations contribute to the variation observed, in the heterogeneity observed in the high viral load group there was an increase in the contribution of the non-synonymous mutations. Thus, this study show that although intrapatient quasispecies heterogeneity is not associated with viral set point in HIV-1 infection, some differences exist between the two groups in the pattern of mutation accumulation.

Amino Acid Substitution↗
PubMed1981

Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription.

Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m(7)GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers, and elongation of the viral mRNAs [Plotch, S. J., Bouloy, M., Ulmanen, L & Krug, R. M. (1980) Cell 23, 847-858]. To identify which viral core protein (nucleocapsid protein, P1, P2, or P3) recognizes the cap 1 structure on the RNA primer, we irradiated (UV) endonuclease reactions carried out by viral cores in the absence of ribonucleoside triphosphates, with a primer RNA labeled in its cap 1 structure with (32)P. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the two basic P proteins, in both one- and two-dimensional gel electrophoresis. This strongly suggests that this crosslinked protein is the viral P3 protein. Competition experiments with unlabeled RNAs containing or lacking a cap 1 structure established that this protein recognizes the cap 1 structure on RNAs. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, we irradiated transcription reactions carried out by viral cores in the presence of [alpha-(32)P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both one-and two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription.

Orthomyxoviridae↗
PubMed1981

Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.

Animals↗
PubMed1998

Control of human immunodeficiency virus type 1 RNA metabolism: role of splice sites and intron sequences in unspliced viral RNA subcellular distribution.

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1 env expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.

Animals↗
PubMed2001

Testing of bulk tank milk from Northern Ireland dairy herds for viral RNA and antibody to bovine viral diarrhoea virus.

Bulk tank milk samples collected from 929 Northern Ireland dairy herds were classified according to their geographical location, somatic cell count and milk yield. Each sample was tested by ELISA for antibody levels to bovine viral diarrhoea virus (BVDV) and the herds were assigned to four groups with increasing antibody levels. In nine herds (1 per cent) no antibodies were detectable in the milk samples (group 1) and they were detectable at low levels in the milk from 90 herds (9.7 per cent, group 2), at moderate levels in 369 herds (39.7 per cent, group 3), and at high levels in the remaining 461 herds (49.6 per cent, group 4). The testing of samples from 90 herds in groups 1 and 2 after an interval of 12 months showed that the annual incidence risk for new infections with BVDV was in the range 0.133 to 0.477. There were significant relationships (P < 0.001) between the mean corrected optical density in the ELISA and herd location and somatic cell count, but not yield; the relationship with somatic cell count was linear (P < 0.01). Forty-three group 3 herds and 49 group 4 herds were selected at random and bulk milk samples were tested for BVDV by reverse transcriptase polymerase chain reaction. None of the group 3 herds was positive, but five group 4 herds were positive.

Animals↗