Semliki Forest virus intracellular RNA: properties of the multi-stranded RNA species and kinetics of positive and negative strand synthesis.

Three replicative forms of RNA (RF I, RF II, and RF III) have been isolated from BHK cells infected with Similiki Forest virus. Using analytical and rate-zonal sedimentation the mol. wt. of these replicative forms were estimated to be 8-5 times 10(6), 5-5 times 10(6) and 3-1 times 10(6) respectively. After continuous labelling from 1 to 6 h post-infection, RFI constituted more than 80% of the total replicative forms. Competition hybridization experiments showed that one strand of RFI was 42S RNA which had opposite (negative) polarity to that found in the virus particle. The positive strand of RFI was 42S RNA. The negative strand of replicative intermediate (RI) was also found to be 42S RNA. No evidence was found for an RI with a 26S negative strand. RFI was shown to contain non-hydrogen bounded poly A at or near the 3' end of the component 42S positive strand. Isolation and analysis of the poly A tract from RFI on an acrylamide gel showed it to be of essentially the same average size as the poly A tract from virus particle RNA. About 30% of the RI molecules contained non-hydrogen bonded poly A. No poly U was detected in either RFI or RI. The kinetics of positive and negative strand synthesis were investigated during virus multiplication. These experiments showed that the rate of negative strand synthesis reaches a maximum 2 1/2 post-infection and thereafter rapidly falls. The rate of positive strand synthesis increases rapidly up to 3 h post-infection and then remains constant for a further 3 to 4 h.     

Mutations in a conserved enteroviral RNA oligonucleotide sequence affect positive strand viral RNA synthesis

Summary.  We showed earlier that a transition mutation U234C, located within the completely conserved 5 nucleotide (nt) tract 5′-CGUUA (nt232–236) in the 5′ non-translated region (NTR) of the coxsackievirus B3 (CVB3) genome, attenuated CVB3 cardiovirulence in mice. To further explore the role of the sequence, we induced two single and one double transversion mutations in the conserved 5mer in a cardiovirulent CVB3 genome. The mutated sites partially or totally reverted to parental wild-type when progeny viruses were passaged at 37 °C, but remained stable when transfection and subsequent passages were performed at 33.5 °C. Viral replication in cell culture was attenuated at 37 °C or 39.5 °C relative to replication at 33.5 °C. While Western blot analysis demonstrated the level of protein translation consistent with virus replication, the ratios of positive to negative strand viral RNA at 37 °C in murine cells demonstrated a 2–5 fold diminution from those measured at 33.5 °C. Mutant CVB3 strains failed to replicate productively when inoculated into mice. The biological data are consistent with an hypothesis that proposes a lesion with primary effects at the level of positive strand viral RNA synthesis that results in attenuation of viral replication at physiologic temperature. Received December 30, 1999 Accepted May 4, 2000     

Hepatitis C virus RNA in patients with chronic hepatitis C

Reviews recent data on the detection of genome and replicative HCV RNA in patients with chronic hepatitis C by PCR and in situ hybridization. Discusses the results of HCV RNA detection in the liver, lymphocytes, serum, and other organs and tissues and notes the relationship between the incidence of RNA and activity of the pathological process. Analyzes the results of HCV RNA detection after IFN treatment. Discusses the role of HCV RNA in the pathogenesis of hepatitis C.     

Translational control in positive strand RNA plant viruses

The great variety of genome organizations means that most plant positive strand viral RNAs differ from the standard 5'-cap/3'-poly(A) structure of eukaryotic mRNAs. The cap and poly(A) tail recruit initiation factors that support the formation of a closed loop mRNA conformation, the state in which translation initiation is most efficient. We review the diverse array of cis-acting sequences present in viral mRNAs that compensate for the absence of a cap, poly(A) tail, or both. We also discuss the cis-acting sequences that control translation strategies that both amplify the coding potential of a genome and regulate the accumulations of viral gene products. Such strategies include leaky scanning initiation of translation of overlapping open reading frames, stop codon readthrough, and ribosomal frameshifting. Finally, future directions for research on the translation of plant positive strand viruses are discussed.     

MHV nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand MHV RNA

We have found that genomic RNA synthesis is inhibited by cycloheximide in cells infected with mouse hepatitis virus, strain A59 (MHV-A59), in agreement with previously published results (Sawicki, S.G. and Sawicki, D.L. (1986) J. Virol, 57, 328-334). In the present study, the fate of the residual genomic RNA synthesized in the presence of cycloheximide was determined. Nearly all of the genomic RNA synthesized in the presence of drug was incorporated into nucleocapsid structures, suggesting that even in the absence of protein synthesis, genomic RNA synthesis and encapsidation are coupled in MHV-infected cells. Sufficient free nucleocapsid N protein was available for this purpose, since the pool of soluble N protein was determined to decay with a half-life of approximately one hour. Negative strand RNA is the template for the synthesis of both genomic and subgenomic positive strand RNA, and would be predicted to accumulate primarily during the early phases of the lytic cycle. In agreement with this prediction, negative strand RNA accumulated during the first 5-6 h of infection, with little additional accumulation occurring over the next 2.5 h. In marked contrast, positive strand RNA increased 5-6-fold over the same 2.5 h period. These results, taken in conjunction with published data, suggest that negative strand RNA is synthesized during the early period of the infectious cycle and is stable in infected cells and also suggest that treatment with cycloheximide at late times does not inhibit positive strand RNA synthesis indirectly by blocking the formation of negative strand templates.     

Charge heterogeneity in polypeptides of negative strand RNA viruses

We surveyed charge heterogeneity in the polypeptides of three enveloped RNA viruses, Sendai virus, influenza virus (WSN strain), and vesicular stomatitis virus (VSV). Isoelectric focusing in polyacrylamide gel was followed by electrophoresis in a second dimension after denaturation of the polypeptides with sodium dodecyl sulfate. Nucleocapsid polypeptides P and NP of Sendai virus exhibited charge heterogeneity which may correspond to various extents of post-translational phosphorylation (R. A. Lamb and P. W. Choppin, 1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology81, 382–397. Nucleocapsid polypeptide N of VSV was more homogeneous, whereas the NP polypeptide of influenza virus appeared to be too basic (isoelectric point greater than 8.0) to be resolved in the isoelectric focusing system employed. Glycosylated envelope polypeptides of all three viruses separated into multiple (3 to 8) acidic species with isoelectric points in the range of 4 to 5 for VSV glycopolypeptide G and 5 to 6 for the glycopolypeptides of Sendai virus and influenza virus. Although some of the heterogeneity in VSV glycopolypeptide G may stem from variations in content of N-acetyl neuraminic acid (NANA), a different basis for the heterogeneity is suggested by failure of extensive neuraminidase treatment to abolish it. Moreover, NANA is removed from the glycopolypeptides of Sendai virus and influenza virus by virus-specified neuraminidases. A possible relationship between content of amino sugars (other than NANA) and charge heterogeneity was suggested by the finding that the amount of radioactive glucosamine incorporated biosynthetically into G of VSV and HN of Sendai virus was greater in the more electropositive species. The functional consequences of this variety of post-translational modifications remain to be determined.     

Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation

Summary. The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5′ untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5′UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5′UTR was necessary for translation; interestingly, when plasmids were used the 5′UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SL_L) that has a structure and stability similar to SL’s found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SL_L had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SL_L or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5′UTR of the SG RNA is required. Further investigation of the differing effects of the SL_L on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SL_Ls could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SL_L only functions in a “cytopathic environment” in which cell translation has been altered. These co-authors contributed equally to the study.     

Hepatitis C virus viremia in HIV-infected individuals with negative HCV antibody tests

Hepatitis C virus (HCV) viremia may occur in persons without detectable HCV antibodies and has been reported in as many as 5.5% of HIV-positive persons. To better characterize serosilent HCV infection, the authors prospectively tested 131 HIV-positive persons and 102 HIV-negative control subjects with diabetes for the presence of HCV antibody (Ab) and HCV RNA. Thirty of 31 HCV Ab-positive (AbP) HIV-positive people tested positive for HCV RNA as did both HCV AbP, HIV-negative control subjects. Similarly, none of the 100 HIV-negative, HCV Ab-negative (AbN) control subjects was HCV RNA positive (p<.001). In contrast, 19 of 100 HIV-positive, HCV AbN persons met stringent criteria for HCV viremia, and 9 of these 19 people were HCV RNA positive when tested by a commercially available HCV RNA detection method. The mean duration of HCV viremia in HCV AbN people was 26.8 months (range, 1-99 months). None of the subjects developed HCV antibody during the study. The HIV-positive, HCV AbP, and RNA-positive group was significantly more likely to have acquired HIV parenterally (p<.001), have higher initial CD4 counts (p=.029), and have higher ALT values than the HCV AbN group (p<.002). In summary, HCV infection appears to occur more frequently among HIV-infected, HCV-seronegative persons than appreciated, especially if HIV acquisition was through sexual as opposed to parenteral risk factors and was associated with a lower initial CD4 count and lower ALT values.     

Cell-free translation ofDrosophila C virus RNA: Identification of a virus protease activity involved in capsid protein synthesis and further studies onin vitro processing of Cricket paralysis virus specified proteins

Summary Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsids precursors of either of the viruses.With 7 Figures     

Hepatitis B virus C gene promoter is under negative regulation.

The regulation of the core promoter of Hepatitis B virus (HBV) was investigated using the chloramphenicol acetyltransferase (CAT) reporter system. Deletional analysis of sequences 5' to the HBV core promoter indicated the presence of a negative regulatory element (NRE) located within a 282-bp BamHI-HincII DNA fragment. The NRE was functional in hepatic as well as nonhepatic cells. Results of in vivo competition experiments suggest a role for cellular transacting repressor protein(s) in the functioning of the NRE. The HBV NRE, positioned 5' to the SV40 early promoter, inhibited the activity of the heterologous promoter in an orientation-independent, but position-dependent manner. These data indicate that the HBV NRE is a silencer element, which functions to downregulate the activity of the core promoter.