Intracellular Viral RNA Levels Research Articles

Persistence of Bovine Viral Diarrhea Virus Is Determined by a Cellular Cofactor of a Viral Autoprotease

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.

Growth kinetics of SARS-coronavirus in Vero E6 cells

Vero E6 cells are commonly used for in vitro studies of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and for antiviral evaluation purposes. A better understanding of the SARS-CoV growth kinetics in Vero E6 cells is crucial to help elucidate the mechanism of antiviral activity of selective antiviral agents. In this study, the growth kinetics of SARS-CoV in Vero E6 cells were studied by quantitation of intra- and extracellular viral RNA load as well as extracellular virus yield at different time points post-infection. At 12 h post-infection, the intracellular viral RNA load was 3 × 10 2-fold higher than at the time of infection, and the extracellular viral RNA load was increased with a factor of 2 × 10 3. Intracellular viral RNA levels started to rise at 6 h post-infection. One hour later (at 7 h post-infection), the levels of extracellular SARS-CoV RNA also began to rise. This was corroborated by the fact that infectious progeny SARS-CoV also first appeared in the supernatant between 6 and 7 h post-infection. At 12 h post-infection, SARS-CoV reached titers in the supernatant of 5.2 × 10 3 CCID 50/ml.

Analysis of spliced and unspliced rous sarcoma virus RNAs early and late after infection of chicken embryo fibroblasts: effect of cell culture conditions

Quantitative determinations of spliced and unspliced viral RNA early after retrovirus infection or after transient transfection with proviral DNA have been difficult because of low intracellular viral RNA levels. In this report we describe conditions for the sensitive and quantitative assay of unspliced viral RNA and spliced mRNAs from avian sarcoma virus-infected and transiently transfected chicken embryo fibroblasts. RNase protection mapping was performed with a tandem 32P-labeled riboprobe consisting of three separate regions complementary to the major 5′ splice site and the two major 3′ splice sites ( env and src). The steady-state levels of viral RNA species were determined as early as 14 hr postinfection; no significant changes were observed in the relative levels of spliced mRNAs or in splice site usage as the infection progressed from 14 to 48 hr. After the cells became morphologically transformed at approximately 72 hr postinfection, the condition of the medium of the transformed cell culture affected the steady-state levels of viral RNAs. Unspliced genomic RNA predominated in the transformed cells at 4.5 to 8 hr after feeding; spliced env mRNA was the major species of RNA at 46 to 103 hr after medium change. In contrast, the steady-state levels of viral RNA in transiently transfected cells or in cells infected with a transformation-defective avian sarcoma virus were not as sensitive to the conditions of medium change. The medium-dependent changes in steady-state levels of viral RNA species may either be caused by a declining transcription rate resulting in the accumulation of the more stable env mRNA relative to the more labile unspliced RNA and src mRNA or to changes in the extent of viral RNA splicing.

Tissue specificity of retrovirus expression in inoculated avian embryos revealed by in situ hybridization to whole-body section

To examine the mechanism of viral tropism in vivo, we injected RAV(1) avian retrovirus into 1-day-old chicken embryos. After 8 days, the majority of the embryos became infected. In situ hybridization to whole embryo sections revealed high levels of intracellular viral RNA, apparently restricted to skeletal muscle cells. The most likely interpretation is that expression of this virus is regulated at the transcriptional level by one or more tissue-specific endogenous factors.

Restriction of murine leukemia proviral gene expression in somatic mouse cell hybrids

Fusion of the N-ecotropic murine leukemia virus (L-MuLV) producing A9HT subline of the mouse L-cell strain with C57B1 lymph node cells gave rise to hybrids with a suppressed production of infectious virus. The hybrids did not release reverse transcriptase or p30 antigen to their media, indicating that there was no detectable production of non-infectious virus. Upon segregation of chromosome 4 of the C57B1 partner cell—as evidenced by the loss of the Gpd-1 a marker—the hybrids resumed production of L-MuLV. The restriction could also be overcome by treatment of the hybrid cells with iododeoxyuridine which induces production of L-MuLV. Intracellular levels of p30 antigen and virus-specific RNA were three- to sevenfold lower in the restricted hybrids than in the A9HT parental cells or in segregating hybrid cell populations that have resumed virus production. Restriction of virus production in hybrid cells is thus correlated with the presence of chromosome 4 from the C57B1 partner cell which appears to affect the levels of intracellular viral RNA and proteins.

RNA synthesis in standard and autointerfered vesicular stomatitis virus infections

The levels of intracellular viral RNA have been measured in BHK-21 cells undergoing standard and autointerfered infections with vesicular stomatitis virus (VSV). The total amount of intracellular nucleocapsid RNA is almost unaffected by autointerference. However, the composition of this class of RNA changes with increasing autointerference: Control cells undergoing standard infections contain only the 140 S, full genomic length nucleocapsid, while autointerfered cells contain, in addition, a smaller nucleocapsid which corresponds in size to that found in the defective interfering (DI) particle. There is a systematic decrease in viral mRNA with increasing autointerference. This decrease in mRNA closely parallels the reduced yield of total progeny particles eventually released from these cells. A causal relationship between the two is proposed. These results are compatible with explanations of autointerference that depend upon a competition between the DI and VSV genomes for the viral replicase.