Selective inhibition of viral protein accumulation in interferon-treated cells; nondiscriminate inhibition of the translation of added viral and cellular messenger RNAs in their extracts

Treatment of monolayers of mouse L cells with 7.5 vesicular stomatitis virus plaque reduction units/ml of mouse interferon (specific activity 2 × 10⁷ NIH mouse reference standard units/mg protein) results in a 95% decrease in the reovirus yield and, as shown earlier, an about 80% decrease in double-stranded and an about 60% decrease in single-stranded reo RNA accumulation in cells infected with 10 plaque-forming units of virus/cell. Interferon at this concentration does not affect the rate of accumulation of host proteins in uninfected or reovirus-infected cells. It inhibits reo virion protein accumulation in infected cells. In control cells the rate of reo virion protein accumulation increases about 2-fold between 10 and 14 hr after infection. In cells treated with interferon it remains at the same low inhibited level. Reo virion proteins were assayed in cell extracts by immunoprecipitation with antisera specific for various size classes or by gel electrophoresis. These results reveal the selective nature of the inhibition of protein accumulation in interferon-treated, virus-infected cells. The effect of treating L cells with interferon was also tested on the capacity of their extracts to translate various mRNAs. The translation of endogenous mRNA, of mRNA in L cell polysomes (added as such) and of polyuridylic acid is at most only slightly inhibited whereas that of added natural mRNAs (including reovirus and encephalomyocarditis virus mRNA, rabbit globin mRNA, and a mixture of L cell mRNAs) is strongly inhibited. The dominant nature of the inhibitor(s) is indicated by the fact that translation of added mRNA is impaired in a mixture of extracts from interferon-treated and from control cells. Thus an effect of interferon treatment of cells is manifested in subcellular systems. However, the selectivity of the effect in vivo, is not well reflected in vitro.     

Biochemical characterization of vesicular stomatitis virus-infected Aedes albopictus cells deprived of methionine

Aedes albopictus cells infected with vesicular stomatitis virus (VSV) and maintained in medium lacking methionine produced 1000-fold less infectious virus than cultures maintained in complete medium. Analysis of viral macromolecular synthesis in cells maintained in the presence of varying concentrations of methionine showed that the reduction in virus yield was directly correlated with a reduction in viral RNA and protein synthesis. Of the viral mRNA which was made in methionine-starved cells the majority was not polysome associated. In contrast, virtually all of the virus mRNA in cells maintained in complete medium was polysome associated. In vitro translation of those mRNAs from methionine-starved cells, which were not polysome associated, indicated that they could be translated in vitro as efficiently as polysome-associated virus mRNA but only if the methylation inhibitor, S-adenosylhomocysteine, was not present. These results strongly suggest that methionine starvation of A. albopictus cells inhibits VSV replication by preventing cap methylation of the viral mRNAs, and thus reducing the efficiency with which they are translanted.     

Studies on the relationship between 5' leader sequences and initiation of translation of adenovirus 2 and simian virus 40 late mRNAs

The question of whether or not 5′ leader sequences might confer characteristic translational properties to the mRNAs to which they are spliced was studied using two complementary experimental situations with respect to the composition of mRNA molecules. Late in adenovirus type 2 (Ad-2) infection of human cells, a common tripartite 5′ leader sequence is spliced to mRNA sequences coding for at least 13 different late viral proteins. This results in different mRNAs with identical 5′ leader sequences. In the second experimental system, identical coding sequences are spliced to different 5′ leader sequences with different caps. Wild-type SV40 VP1 mRNA (16 S) contains a 5′ leader sequence coded for at position 72–76 on the SV40 genome; in the SV40 mutant dl-808 part of the sequences in this region, including the sequence coding for cap, is deleted. The relative ability of these mRNAs to direct incorporation of radioactive amino acids into their respective polypeptide products was assayed in vivo under conditions of hypertonic initiation block (HIB) or in the presence of low concentrations of cycloheximide. Messenger RNAs with identical 5′ leader sequences were observed to exhibit very different responses in both experimental situations. Conversely, similar responses to HIB were observed for VP1 mRNAs with different 5′ leader sequences. It thus appears that the presence of specific sequences per se between the 5′ cap and the initiation codon does not alone confer characteristic initiation properties as measured indirectly by perturbation of the in vivo environment.     

Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species

Inhibition of HSV-1 DNA synthesis with either arabinosyladenine plus the adenosine deaminase inhibitor pentostatin, or with arabinosylthymine, showed a viral mRNA population identical to that seen prior to viral DNA replication (early) by the criteria of quantitative hybridization, specific mRNA species identifiable by Southern blot hybridization of size-fractionated RNA, and migration of polypeptides resolved by in vitro translation of purified viral mRNA. The amount of viral mRNA associated with infected cell polyribosomes was determined by quantitative DNA excess solution hybridization. At 2 hr postinfection (p.i.) (before viral DNA synthesis) and in drug-treated cells at 6 hr p.i., the majority of the polyadenylated RNA was cell specific with some virus-specific RNA detectable. In contrast, at 6 hr p.i., in the absence of drugs (during maximum viral DNA synthesis), nearly all the polyadenylated polyribosomal RNA was viral. Blot hybridization of size-fractionated viral RNA confirmed several specific differences between the viral mRNA species occurring before and after HSV-1 DNA synthesis, which have been reported previously from this laboratory. These differences also were reflected in the in vitro translation products encoded by the viral mRNAs. The mRNA species and the encoded polypeptides that were present in the absence of viral DNA synthesis are a subset of those viral mRNA species and polypeptides expressed in the presence of viral DNA synthesis. The viral mRNA species fall into several groups based on their relative abundance at various times of infection. These data suggest that, in the normal virus infection cycle, the onset of viral DNA synthesis is necessary for normal expression of later viral genes.     

Synthesis of VSV RNPs in vitro by cellular VSV RNPs added to uninfected HeLa cell extracts: VSV protein requirements for replication in vitro

Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited.     

TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements

In mammalian cells, mRNAs with AU-rich elements (AREs) are targeted for translational silencing and rapid degradation. Here we present evidence that in human cells the proteins Tristetraprolin (TTP) and BRF-1 deliver ARE-mRNAs to processing bodies (PBs), cytoplasmic assemblies of mRNAs, and associated factors that promote translational silencing and mRNA decay. First, depletion of endogenous TTP and BRF proteins, or overexpression of dominant-negative mutant TTP proteins, impairs the localization of reporter ARE-mRNAs in PBs. Second, TTP and BRF-1 localize tethered mRNAs to PBs. Third, TTP can nucleate PB formation on untranslated mRNAs even when other mRNAs are trapped in polysomes by cycloheximide treatment. ARE-mRNA localization in PBs is mediated by the TTP N- and C-terminal domains and occurs downstream from mRNA polysome release, which in itself is not sufficient for mRNA PB localization. The accumulation of ARE-mRNAs in PBs is strongly enhanced when the mRNA decay machinery is rendered limiting by mRNA decay enzyme depletion or TTP/BRF-1 overexpression. Based on these observations, we propose that the PB functions as a reservoir that sequesters ARE-mRNAs from polysomes, thereby silencing ARE-mRNA function even when mRNA decay is delayed. This function of the PB can likely be extended to other mRNA silencing pathways, such as those mediated by microRNAs, premature termination codons, and mRNA deadenylation.     

Nontranslated cellular mRNAs are associated with the cytoskeletal framework in influenza virus or adenovirus infected cells

In an effort to understand the molecular mechanisms underlying the selective shutoff of host protein synthesis in influenza virus and adenovirus infected cells, we analyzed the subcellular location of representative cellular and viral mRNAs. Earlier work has shown that the majority of cellular mRNAs remain polysome associated after infection by either virus and that both the initiation and elongation steps of host protein synthesis were blocked in infected cells (M. G. Katze, D. DeCorato, and R. M. Krug, J. Virol., 60, 1027-1039, 1986). The present study was undertaken to test whether these cellular mRNAs were rendered nontranslatable during infection as a result of their dissociation from the cytoskeleton framework. HeLa cells were fractionated into subcellular components by first gently disrupting the cells with Triton X-100 yielding the soluble fraction (SOL); the cytoskeleton (CSK) fraction was obtained from the Triton insoluble material by the double detergent treatment of Tween-40 and sodium deoxycholate. In uninfected cells the majority of host mRNAs were associated with polysomes which were exclusively bound to the CSK as would be expected of actively translated mRNAs. The cellular mRNAs also remained almost totally associated with the cytoskeleton in adenovirus and influenza virus infected cells despite the fact that these mRNAs are not translated during infection. Indeed, the host mRNAs and the efficiently translated viral mRNAs were CSK associated to the same extent. In contrast to the adenovirus and influenza systems, significant amounts of cellular mRNAs were dissociated from the CSK and found in the SOL fraction of poliovirus infected cells as others have reported. In accordance with the biochemical analysis, morphological studies utilizing electron microscopy revealed that the cytoskeleton remained relatively intact during adenovirus and influenza infection but was substantially reorganized in poliovirus infected cells. We conclude that translational regulatory events are likely different in the poliovirus system and that cytoskeletal association of mRNAs may be required but is not sufficient for efficient mRNA translation during adenovirus or influenza virus infection.     

Mechanisms of regulation of ferritin synthesis in rat liver during experimental inflammation

Liver slices of turpentine-treated rats were incubated in vitro and used as a model to study synthesis and secretion of proteins during the acute-phase response. The synthesis and secretion of typical acute-phase proteins increased after treatment. Similarly, ferritin increased at 24-48 hr after treatment. Serum ferritin showed a slight and transient increase at 6 hr; however, no ferritin was detectable in liver slices medium, indicating no or negligible secretion by this tissue. Northern blot analysis of RNA extracted from total liver homogenate and from free and membrane-bound polyribosomes revealed that turpentine treatment stimulates ferritin synthesis at the translational level, possibly increasing the amount of ferritin mRNA on membrane-bound polysomes.     

A quantitative sucrose gradient analysis of the translational activity of 18 mRNA species in testes from adult mice.

Sucrose gradients have been widely used to study the translational activity of mRNA species in meiotic and haploid spermatogenic cells in mammals. Unfortunately, the results of these studies have been very inconsistent. The purpose of the present study was to obtain accurate and reproducible measurements of the translational activity of a large number of testicular mRNA in sucrose gradients. Extracts of adult testes and cultured seminiferous tubules were sedimented on sucrose gradients, and the distribution of 18 mRNA species was quantified by phosphoimaging. The proportions of various mRNA species sedimenting with polysomes in meiotic and haploid cells (approximately 6-74%) is less than typical of efficiently translated mRNAs (85-90%), demonstrating that the initiation of translation of virtually all mRNA species is at least partially inhibited and that the extent of inhibition is mRNA-specific. Most mRNA species in meiotic and early haploid spermatogenic cells are translated on polysomes in which the ribosome spacing is somewhat wider than in somatic cells, 100-150 verses 80-100 bases. However, the ribosome spacing on protamine mRNAs is unusually close (40-50 bases), and the spacing on poly(A) binding protein mRNA is unusually wide (212-272 bases), thus suggesting that the rate of translational initiation, termination and/or elongation is regulated on translationally active forms of certain mRNA.     

Vesicular stomatitis virus pseudotypes produced by cells abortively infected or transformed by human cytomegalovirus

Summary Pseudotypes of vesicular stomatitis virus (VSV) and human cytomegalovirus (HCMV) were produced by normal hamster cells abortively infected with HCMV and superinfected by VSV at a certain stage of abortive HCMV infection. Hamster cells transformedin vitro by HCMV (87-TRH-5 and CX-90-3B cells) also produce VSV (HCMV) pseudotypes after infection of the cells by VSV, but the same cells after passagein vivo (TSC-1, TSC-2 cells) do not.With 3 Figures     

Studies on the in vivo and in vitro messenger RNA species of vesicular stomatitis virus.

The messenger RNA species isolated from vesicular stomatitis virus (VSV)-infected cells were compared with those synthesized in vitro by the virion-associated RNA polymerase. In both cases, the messenger RNAs were complementary to the genome RNA and consisted of four size-classes that had sedimentation coefficients of 31 S, 17 S, 14.5 S and 12 S in sucrose velocity gradients and that comigrated in denaturing polyacrylamide gels. Competition of the 31 S or 12–18 S mRNA species in annealing with the VSV genome RNA showed that the RNA species synthesized in vitro were identical in base sequence to the corresponding in vivo VSV-RNAs. Each of the RNA species contained poly(A) sequences and bound to oligo(dT)-cellulose to a similar extent. While the in vivo 12–18 S mRNAs contained poly(A) sequences of very heterogeneous length, the corresponding in vitro mRNA species possessed a unique length of poly(A) of about 200 nucleotides.     

Restriction of vesicular stomatitis virus in a nonpermissive rabbit cell line is at the level of protein synthesis

Replication of vesicular stomatitis virus (VSV) is restricted in a line of rabbit cornea (RC-60) cells; less than one infectious particle is produced per infected cell. We show that VSV is blocked at the level of viral-specific protein synthesis. VSV proteins are synthesized early in infection but the rate of VSV protein synthesis declines rapidly as infection proceeds. At times when synthesis of VSV proteins is barely detectable, VSV mRNA is produced and polyribosome structures are present. The VSV mRNA recovered from the polysome region directs the synthesis of VSV proteins in an in vitro reticulocyte translation system. This suggests that protein synthesis is blocked at some step beyond the level of initiation possibly at the level of elongation. Coinfection with vaccinia virus converts RC-60 cells to a permissive host. In contrast to the abortive infection with VSV alone, VSV proteins are synthesized throughout the replication cycle in doubly infected cells. Vaccinia supplies a product essential for sustained protein synthesis in the abortive system. We have confirmed that the replication of genome length 42 S RNA does not occur at late times in the abortive infection. This lack of 42 S RNA replication is explained by the shut-off of VSV protein synthesis, since continuous protein synthesis is required for the replication of VSV 42 S RNA.