Viral interference in HIV-1 infected cells

The study of viral interference in HIV-1 infected cells has revealed several different means whereby infected cells resist superinfection. The most familiar of these, down-modulation of cellular receptors for virus, can be accomplished through the independent action of at least three HIV-1 proteins. Both the principal viral receptor CD4 and the chemokine receptors which serve as co-receptors are subject to down-modulation as a consequence of infection. Elucidation of the specificity of co-receptor utilisation by HIV-1 strains is an exciting, ongoing task which has opened new avenues to the understanding of viral replication and pathogenesis. Novel routes to resistance to superinfection have been discovered during HIV-1 infection and their investigation may reveal new pathways to control HIV-1 and the loss of immunological function with AIDS. Copyright © 1998 John Wiley & Sons, Ltd.     

Viral messenger RNAs in six lines of adenovirus-transformed cells

We have compared the proteins translated in vitro using virus-specific mRNA from six lines of adenovirus-transformed cells (F4, F17, F19, REM, T2C4, and 293) with those made using RNA from productively infected cells. The mRNAs for proteins E1A-58K, 54K, 48K, and 42K, and for E1B-15K, were detected in all cell lines and for E1B-57K in at least five of the lines. Neither of the minor E1A mRNAs (for proteins 25K and 28K) found under specific conditions in infected cells were found in any transformed cell line. Two cell lines, F4 and T2C4, contained small amounts of other mRNAs encoded within region E1B including in the latter case one not detected in infected cells. The lines F4, REM, and T2C4 contain at least some E4 mRNAs, again including in the latter case one not detected in infected cells. T2C4 cells also contain E3 mRNAs, E2A-72K protein mRNA, and two E2B mRNAs, although the E2B proteins are not identical to any found in infected cells. The F19 and T2C4 lines express mRNAs encoded between 11 and 17 map units, the “IVa₂ region,” but do not include the mRNA for polypeptide IVa₂ itself. Some early mRNAs, including the “immediate early” mRNAs, have not been detected in any of these lines of transformed cells: nor did the cell lines contain any mRNAs found exclusively at late times during productive infection. Comparison of these results with the properties of the cells tentatively suggest a correlation (although not necessarily causal) between the expression of additional viral genes and increased tumorigenicity.     

Viral genome synthesis in BHK 21 cells persistently infected with Sendai virus

We have carried out daily analysis of the viral RNA species replicated by nucleocapsids present in the cytoplasm of two different BHK 21 cell lines persistently infected with Sendai virus. At each day of analysis (both before and after crises of cytopathology, and during long periods of stable growth) DI particle size RNA was the major species of viral RNA replicated within these nucleocapsids. The molar ratio of DI RNA to standard virus (50 S) RNA species within intracellular nucleocapsids was always extremely high (from 10/1 to 340/1). Thus, there is no evidence for cyclic variations in relative predominance of standard virus and DI nucleocapsids in populations of these persistently infected cells, although “cycling” could occur at the individual cell level at infrequent intervals. Furthermore, challenge of persistently infected cultures with standard Sendai virus or incubation at 33° also failed to significantly decrease the ratio of DI nucleocapsids to standard virus nucleocapsids. It was also found that multiple DI RNA species are present (most are not resolvable by sucrose gradient analysis), and that these evolve with time—some arising and increasing with time, while others are outcompeted. No selective advantage for DI RNA of any particular size was observed. Finally, by measuring the total amount of nucleocapsids present in these persistently infected cells (as estimated by level of NP protein in purified nucleocapsids), and the degree of synthesis of viral nucleocapsid (as determined by [³H]uridine incorporation in the presence of actinomycin D), we found that the rate of viral genome replication was 30- to 40-fold lower in the persistently infected cells than it was in St acutely infected cells. These findings may indicate that the accumulated nucleocapsids in persistently infected cells are built up over a period of many weeks of slow synthesis, although turnover times for NP protein and RNA of nucleocapsids have not yet been determined in persistently infected cells.     

Viral nucleic acid synthesis in HSV infected neural cells

Summary HSV-1 replication and synthesis of viral DNA and RNA have been examined in gliomas of human (COX) and rat origin (C6) and in mouse neuroblastomas (D2). COX cells fully support HSV-1 replication and show patterns of viral DNA and RNA synthesis similar to those seen in continuous line cells. HSV-1 also grows to high titers in D2 cells but without concomitant high levels of viral DNA and RNA synthesis in the infected cells. Finally, HSV-1 established a persistent infection in C6 cells. Viral mRNA and DNA synthesis could not be detected in these cultures. At cycles of approximately 15–20 days, the persistently infected cultures exhibited massive CPE and relatively high production of infectious HSV.With 3 Figures     

Viral products in cells infected with Vesicular Stomatitis virus and superinfected with Rous Sarcoma virus

Summary In cells infected with Vesicular Stomatitis virus (VSV) ts 1026 and superinfected with Rous Sarcoma virus (RSV) synthesis of vsrc mRNA and RSV env mRNA decreases. In these cells post-translational processing of RSV precursor proteins is impaired and small amounts of VSV antigens are detected.     

Studies on virus-specific nucleic acids synthesized in vertebrate and mosquito cells infected with flaviviruses

Virus-specific RNA molecules synthesized in BHK 21 vertebrate cells and in Aedes albopictus mosquito cells infected with the flaviviruses Uganda S (US) or West Nile (WN) have been characterized. A single-stranded (ss) RNA of plus polarity sedimenting at about 42 S was present in the virus particles. 42 S plus strand RNA was also the predominant species of virus-specific ss RNA accumulating in infected cells of both vertebrate and insect origin. No similarity was detected between the large oligonucleotides generated by ribonuclease T₁ from WN virus and US virus-specific 42 S plus strand RNA, respectively. No poly(A) sequences are present in either the US virus or the WN virus-specific 42 S plus strand RNA molecules synthesized in BHK cells. The 42 S plus strand RNA molecules present in WN virus-infected BHK cells do contain, however, a “cap” structure m⁷GpppAmpN₁, and in some of these molecules, a further methyl group is introduced, giving rise to the “cap” structure m⁷GpppAmpN₁mpN₂. These “caps” are also present on the 42 S RNA of WN virus particles synthesized in BHK cells. In addition to the 42 S RNA, virus-specific ss RNA of low molecular weight (LMW-RNA) was detected in all virus-cell systems analyzed. A single species of LMW-RNA of 5 × 10⁴ daltons apparent molecular weight was present in US virus-infected vertebrate and insect cells. Two LMW-RNA species of about 6.5 × 10⁴ daltons (WM LMW-1 RNA) and 4.2 × 10⁴ daltons (WN LMW-2 RNA) molecular weight, respectively, were isolated from WN virus-infected BHK cells. Only the larger of these was detected in WN virus-infected insect cells. The LMW-1 RNA present in WN virus-infected BHK cells has been characterized in somewhat more detail. It contains virus-specific RNA sequences of plus strand polarity, is not “capped”, and does not contain a poly(A) sequence. None of the single-stranded, virus-specific RNA molecules synthesized in either vertebrate or mosquito cells bound to oligo(dT)-cellulose. Only a single species of virus-specific RNA containing minus strand sequences was detected in WN virus-infected BHK cells. This RNA was of genome size and was present as part of a double-stranded RNA complex containing 42 S RNA of both plus and minus polarity.     

Comparison of messenger RNAs induced in cells infected with each member of the morbillivirus group

Virus-specific mRNAs radiolabelled with [32P]orthophosphate in the presence of actinomycin D were extracted from the cytoplasm of Vero cells infected with each of the known morbilliviruses: measles virus, canine distemper virus, rinderpest virus, and peste des petits ruminants virus. When analysed on denaturing agarose-formaldehyde gels the major RNA species from all viruses in the group were identical, except for canine distemper virus where one of the virus-specific mRNAs (mRNA 5), which probably codes for the virus haemagglutinin (S.E.H. Russell, D. K. Clarke, E. M. Hoey, B. K. Rima, S. J. Martin, J. Gen. Virol. 66, 433-441 (1985], was significantly smaller than the corresponding mRNA induced by the other viruses. Plasmid DNA containing a virus-specific insert, representing greater than 98% of the gene derived from the P-protein mRNA of canine distemper virus, showed significant cross-hybridisation with all the other members of the morbillivirus group.     

Viral noncoding RNAs: more surprises

Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles—including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation—have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action.     

The metabolism of host RNAs in cells infected by an adenovirus E4 mutant

Mutants of adenovirus type 5 (Ad5) that lack early region 4 (E4) are defective in the expression of viral late genes. E4 mutants exhibit dramatically reduced levels of both cytoplasmic and nuclear viral late RNAs compared to wild-type virus, due principally to reduced stability of unprocessed viral late RNA in the nucleus of mutant-infected cells. To determine whether E4 products also affect the metabolism of host RNAs in infected cells, steady-state levels of beta-actin RNA and triose phosphate isomerase (TPI) RNA were measured in the cytoplasms and nuclei of HeLa cells infected by either wild-type Ad5 or the E4 deletion mutant H5dl1004, and were compared to levels in uninfected HeLa cells. S1 nuclease analyses revealed only slight reductions in beta-actin mRNA levels in the cytoplasm and in levels of spliced and unspliced beta-actin RNA in the nucleus of cells infected by either Ad5 or H5dl1004. RNase protection analyses showed that cytoplasmic TPI RNA levels were not affected by infection of HeLa cells with either Ad5 or H5dl1004. Steady-state levels of nuclear TPI RNA, both spliced and unspliced, were slightly reduced in cells infected by wild-type virus but not in HeLa cells infected by H5dl1004. These results indicate that the reduced stability of RNA in HeLa cells infected by E4 mutants is a virus-specific phenotype which does not extend to host cell RNAs.     

Characterization of RNAs specific to avocado sunblotch viroid synthesized in vitro by a cell-free system from infected avocado leaves.

Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell-free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200 micrograms/ml of the drug, which typically inhibit RNA polymerase II and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polycrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis.     

Vaccinia virus antigens on the plasma membrane of infected cells. I. Viral antigens transferred from infecting virus particles and synthesized after infection

SDS-polyacrylamide gel electrophoretic analysis of plasma membranes prepared from L cells infected with radioiodinated vaccinia virus particles showed that at 2.0 hr postinfection, 125I-labeled virion polypeptides with molecular weights of 58K-60K, 32K-34K, 17K, and 12K-14K were associated with infected cell plasma membranes. By 4 hr postinfection, only the 32- to 34-kDa polypeptide, derived from infecting virus particles, could be detected on infected cell surfaces. A variety of techniques were applied to analyzing purified plasma cell membranes to define the viral antigens expressed on cell surfaces after infection, including (a) surface radioiodination of infected cells; (b) immune or Western blotting; (c) specific immunoprecipitation of viral proteins present in nonionic detergent extracts of membranes purified from [35S]methionine-labeled, virus-infected cells. It was determined that vaccinia virus-specified polypeptides with molecular weights of 78K-82K, 65K, 50K, 42K-45K, 35K-37K, 32K-34K, 30K, 20K, and 17K-18K were expressed by 3 hr postadsorption, on the plasma membranes of infected cells and were accessible to binding by exogenous antiviral antibodies. Viral antigens with molecular weights similar to those expressed on cell surfaces were secreted or shed from infected cells and could be detected in the medium harvested from virus-infected mouse L-cell cultures.