Black beetle virus: messenger for protein B is a subgenomic viral RNA

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein α (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected Drosophila cells. Two of these proteins, A and α, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and α cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.     

Dexamethasone Induction of the Intracellular RNAs of Mouse Mammary Tumor Virus

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5′ and 3′ termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.     

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.     

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.     

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3′ end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.     

Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA.

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.     

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.     

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.     

The Plant Host Can Affect the Encapsidation of Brome Mosaic Virus (BMV) RNA: BMV Virions Are Surprisingly Heterogeneous

Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses.     

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.     

Synthesis of genomic and subgenomic RNA in mosquito cells infected with two Sindbis virus nsP4 mutants: influence of intracellular nucleoside triphosphate concentrations

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.     

MicroRNAs and Unusual Small RNAs Discovered in Kaposi's Sarcoma-Associated Herpesvirus Virions

It is widely held that any given virus uses only one type of nucleic acid for genetic information storage. However, this consensus has been challenged slightly by several recent studies showing that many RNA species are present within a range of DNA viruses that include Kaposi''s sarcoma-associated herpesvirus (KSHV). RNAs extracted from purified DNA virus particles exhibit great diversity in terms of length, abundance, temporal expression, cellular localization, and coding capacity during viral infection. In addition to known RNA species, the current study showed that small regulatory RNAs were present in KSHV virions. A large number of viral and cellular microRNAs (miRNAs), as well as unusual small RNAs (usRNAs), were detected in KSHV virions by using deep sequencing. Both viral and host miRNAs detected in small RNAs extracted from KSHV virions were further shown to colocalize with KSHV virions directly by in situ hybridization (ISH)-electron microscopy (EM) (ISH-EM). Some of these miRNAs were differentially present in the host cells and KSHV virions, suggesting that they are not randomly present in KSHV virions. The virional miRNAs could be transported into host cells, and they are biologically functional during de novo viral infection. Our study revealed miRNAs and usRNAs as a novel group of components in KSHV virions.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]