Accumulation Of Viral mRNA Research Articles

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein

The distribution of the adenovirus early region 1B 55-kDa protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral protein encoded by early region 4 (E4-34kDa). This protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa protein was not required for the close association of the early region 2A DNA binding protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.

Role of soluble cytokines on the restricted replication of poliovirus in the monocytic U937 cell line

Poliovirus infection of monocytic U937 cells is characterized by an incomplete shut-off of host protein synthesis occurring at late times postinfection and by a slow accumulation of viral mRNA which is not efficiently translated. By using exogenous cytokines, neutralizing antibodies to different cytokines and specific cDNA probes, we have studied the involvement of soluble factors on the restricted replication of poliovirus in U937 cells. Our results show that both IFN-γ and TNF-α mRNA levels increased in U937 cells upon poliovirus infection. Moreover, treatment of infected U937 cells with antibodies to IFN-γ induced a remarkably stronger shut-off of host protein synthesis than that found in control cultures. A role of IFN-γ in protecting the infected cells from an extensive blockade of host mRNA translation is proposed.

Restriction of poliovirus RNA translation in a human monocytic cell line

The infection of the human monocytic cell line U-937 by poliovirus was characterized by a low level of virus production and a slow progression of the cytopathic effect. Infection took place in greater than 99% of the cells as revealed by a limiting dilution assay. No viral protein synthesis was evident in the infected U-937 cells when analyzed by polyacrylamide gel electrophoresis. However, a low level of poliovirus RNA translation was detected by immunofluorescence analysis using a mixture of polyclonal antisera against non-structural proteins. Although there was only a low level of viral protein synthesis, a gradual accumulation of viral mRNA took place in U-937 cells as revealed by RNA blot analysis. Similar results were obtained when the erythroleukemic cell line K-562 was used as a host cell for poliovirus. RNA extracted from infected U-937 cells was efficiently translated in rabbit reticulocyte extracts giving rise to a pattern of viral polypeptides similar to that detected when virion-purified RNA was the template used for the in vitro translation assay, suggesting that the poliovirus RNA present in infected U-937 cells is functional. The existence in U-937 cells of a discriminatory mechanism which differentially interferes with poliovirus RNA translation is discussed.

Trans-acting regulation of bovine leukemia virus mRNA processing.

Bovine leukemia virus (BLV) and the human T-cell leukemia virus types I and II comprise a unique retrovirus subfamily which has evolved complex strategies for the regulation of gene expression. A transcriptional control circuit has been characterized in both human and bovine systems in which cis-acting promoter control elements are responsive to trans-acting factors encoded in the pX region of the virus. The BLV pX mRNA encoding the transcriptional trans-acting factor is translated in an alternate reading frame to produce an 18-kilodalton nuclear phosphoprotein, p18. A function for this protein was revealed in cotransfection experiments using mutated BLV proviruses in combination with pX expression plasmids. These experiments indicated that p18 was required for the accumulation of viral mRNAs representing full-length (genomic) and single-spliced (env) transcripts. In contrast, synthesis of the double-spliced pX mRNA was not influenced by p18 expression. Large regional deletions and substitutions of provirus sequences localized elements essential for p18 regulation to the 3' long terminal repeat. Furthermore, sequences within a 250-nucleotide region between the AATAAA signal and poly(A) site were found to be essential for efficient virus mRNA 3'-end processing and response to p18 regulation.

Priming of influenza mRNA transcription is inhibited in CHO cells treated with the methylation inhibitor, Neplanocin A

Chinese hamster ovary cells were pretreated with Neplanocin A, a potent inhibitor of RNA methylation. Analysis of polyadenylated RNA from treated cells by high-pressure liquid chromatography revealed marked decreases of 2′- O-methylation within mRNA cap structures and of internal N 6-methyladenosine residues. In these Neplanocin A-treated cells, influenza viral mRNA accumulation was virtually abolished. Cellular RNA from Neplanocin A-treated cells was substantially less efficient than RNA from control cells in priming cell-free influenza transcription reactions. These results suggest that the observed inhibition of influenza virus replication is due at least in part to impaired recognition of undermethylated cellular mRNA cap structures by the influenza polymerase complex.

Accumulation of herpes simplex virus type 1 RNAs of different kinetic classes in the cytoplasm of infected cells.

We have analyzed the accumulation of herpes simplex virus type 1 RNA of the immediate early (IE; infected cell polypeptide types 4 and 0 [ICP-4 and ICP-0]), early (thymidine kinase), and early late (ICP-5) kinetic classes in the cytoplasm of infected cells in the presence of anisomycin, canavanine, or phosphonoacetic acid and in the course of a normal infection. IE RNAs were overproduced and were the only class of transcript detected in anisomycin-blocked cells. Phosphonoacetic acid treatment resulted in overaccumulation of early RNAs and underaccumulation of early late RNAs. Although low-stringency canavanine treatment resulted in accumulation of RNA from all kinetic classes, high-stringency conditions restricted accumulation of herpes simplex virus type 1 RNAs to the IE class. More importantly, the IE RNAs for ICP-4 and ICP-0 accumulated to a lesser extent under high-stringency canavanine conditions compared with their accumulation in anisomycin-treated cells. Therefore, the absence of newly synthesized viral proteins (anisomysin treatment) and the presence of analog proteins (stringent canavanine treatment) have different consequences with regard to the accumulation of these two IE RNAs. The kinetics of cytoplasmic accumulation for these RNAs was different for each class of RNA. The IE RNAs were detectable at 1 h postinfection and reached a maximum accumulation at ca. 3 h postinfection. The IE RNAs for both ICP-4 and ICP-0 persisted at late times of infection; however, they differed in that the RNA for ICP-4 remained at relatively low levels and the RNA for ICP-0 remained at relatively high levels as compared with their peak levels of accumulation. The 1.4-kilobase RNA for the herpes simplex virus type 1 thymidine kinase was detected by 2 h, with maximum accumulation occurring at ca. 5 h postinfection. After the peak of accumulation, the amount of thymidine kinase RNA declined rapidly from 8 to 14 h postinfection. The early late RNA for ICP-5 was detected between 2 and 3 h, after which accumulation increased to a peak between 8 and 10 h postinfection. The level of ICP-5 RNA remained at close to the peak level until 14 h postinfection. We also compared the accumulation of viral mRNAs in the cytoplasm with the rates of synthesis of their respective polypeptides. Our results suggest that translational controls may be involved in the regulation of IE genes but not early or late genes.

Interferon pretreatment inhibits simian virus 40 infections by blocking the onset of early transcription

CV-1 cells treated with human leukocyte interferon before infection with simian virus 40 (SV40) are protected from virus-induced cytopathic effects. Accumulation of viral mRNA is inhibited markedly. If T antigen is present in interferon-treated cells before infection, viral DNA replication is inhibited only slightly. These results show that many SV40 virions reach the nucleus in a form which can be replicated but not transcribed. Accumulation of SV40 mRNA is not inhibited in cells treated with interferon after infection. Thus the block mediated by interferon is specific for the onset of transcription. Infections with SV40 DNA rather than virions are insensitive to treatment with interferon, showing that the inhibition involves an interaction between viral nucleoprotein complexes and cellular factors.

Control of adenovirus early gene expression: accumulation of viral mRNA after infection of transformed cells.

We studied the accumulation of viral mRNA in the presence of inhibitors of protein synthesis in an adenovirus type 5-transformed cell line (line 293 cells). An analysis of the endogenous viral mRNA's and proteins revealed that only early regions 1A and 1B were expressed in uninfected 293 cells. However, viral mRNA's from early regions 2, 3, and 4, as well as mRNA's from early regions 1A and 1B, accumulated in 293 cells after infection with adenovirus type 2. Cells treated with anisomycin before infection showed a drastic enhancement of mRNA from early region 4 compared with drug-free controls, This increase in viral mRNA was detected by using filter hybridization, S1 endonuclease mapping, and in vitro translation. The rate of transcription of early region 4 nuclear RNA also increased significantly in the presence of anisomycin. In contrast to the results with early region 4, the levels of cytoplasmic mRNA's from early regions 2 and 3 did not increase in cells treated with inhibitors. These results suggested that multiple virus encoded controls operate on the early regions of the adenovirus genome.

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, "P." We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA. A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.