HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms

In addition to genomic RNA, HIV-1 particles package cellular and spliced viral RNAs. In order to determine the encapsidation mechanisms of these RNAs, we determined the packaging efficiencies and specificities of genomic RNA, singly and fully spliced HIV mRNAs and different host RNAs species: 7SL RNA, U6 snRNA and GAPDH mRNA using RT-QPCR. Except GAPDH mRNA, all RNAs are selectively encapsidated. Singly spliced RNAs, harboring the Rev-responsible element, and fully spliced viral RNAs, which do not contain this motif, are enriched in virions to similar levels, even though they are exported from the nucleus by different routes. Deletions of key motifs (SL1 and/or SL3) of the packaging signal of genomic RNA indicate that HIV and host RNAs are encapsidated through independent mechanisms, while genomic and spliced viral RNA compete for the same trans-acting factor due to the presence of the 5′ common exon containing the TAR, poly(A) and U5-PBS hairpins. Surprisingly, the RNA dimerization initiation site (DIS/SL1) appears to be the main packaging determinant of genomic RNA, but is not involved in packaging of spliced viral RNAs, suggesting a functional interaction with intronic sequences. Active and selective packaging of host and spliced viral RNAs provide new potential functions to these RNAs in the early stages of the virus life cycle.     

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.     

Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA

The purified p12 phosphoprotein of Rauscher murine leukemia virus was fractionated by ion exchange chromatography into subpopulations of molecules containing different amounts of covalently linked phosphate. Of the various phosphorylated forms of p12 protein purified from virions, only a species containing relatively little phosphate can bind in vitro to purified homologous 70S viral RNA. Using ultraviolet irradiation to stabilize ribonucleoprotein complexes in intact virions, the same molecular species of p12 phosprotein can be isolated in close association with the 70S viral genome. The results show that phosphorylation of type C viral p12 proteins influences the extent, but not the specificity, of their interaction with homologous viral RNA.     

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates the replication of Bamboo mosaic virus and its associated satellite RNA

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3' untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ～35 and ～55 kDa were specifically cross-linked with RNA elements, whereupon the ～35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3' UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3' UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.     

Encapsidation of turnip crinkle virus is defined by a specific packaging signal and RNA size.

A protoplast infection assay has been used to reliably examine the viral RNA encapsidation of turnip crinkle virus (TCV). Analysis of the encapsidation of various mutant viral RNAs revealed that a 186-nucleotide (nt) region at the 3' end of the coat protein (CP) gene, with a bulged hairpin loop of 28 nt as its most essential element, was indispensable for TCV RNA encapsidation. When RNA fragments containing the 186-nt region were used to replace the CP gene of a different virus, tomato bushy stunt virus, the resulting chimeric viral RNAs were encapsidated into TCV virions. Furthermore, analysis of the encapsidated chimeric RNA species established that the RNA size was an important determinant of the TCV assembly process.     

Structural components of the arenavirus Pichinde.

Purified Pichinde virions grown in monolayers of BHK-21 cells were found to contain three major species of virion proteins as described previously (Ramos et al., J. Virol. 10:661-667, 1972). Two of the proteins were glycosylated (G1, molecular weight = 64,000; G2, molecular weight = 38,000) and were present in similar proportions on the outer surface of the virions. A third protein (N, molecular weight = 66,000) was not glycosylated and, in association with the viral RNA species, was the major protein component of the viral nucleocapsids. An estimate of the approximate number of molecules of these three major proteins per virion was made. Minor amounts of other proteins were also routinely observed in Pichinde virus preparations. None of the three major protein species were phosphorylated to any significant exten, nor did they contain sulfated components. Two virion RNA species (L and S), but no 18S rRNA species, were detected in Pichinde virus preparations obtained from infected BHK-21 cells.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3'' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.     

Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants

The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex of Tomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used an in vitro TBSV replication assay based on recombinant p33 and p92(pol) viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92(pol) viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.     

Protein species of the parvovirus minute virus of mice strain MVMp: involvement of phosphorylated VP-2 subtypes in viral morphogenesis.

The pattern of induced protein species of the prototype strain of the parvovirus minute virus of mice was determined in permissive A9 mouse fibroblast cells by high-resolution two-dimensional gel electrophoresis. Identities of the viral proteins in the gels were assigned by probing two-dimensional blots with antisera raised against either purified capsids (recognizing VP-1 and VP-2) or specific coding regions of the nonstructural proteins (NS-1 and NS-2) expressed as beta-galactosidase fusion products in bacteria. All viral proteins showed posttranslational modifications, phosphate being a common substituent. The NS-1 protein migrated as a basic polypeptide in the pI range of 7.4 to 7.8 with multiple stages of modification and as a likely minor but hyperphosphorylated component in the neutral region of the gel. The NS-2 isoforms were resolved at a pI value close to 5.5 as three groups of unevenly phosphorylated polypeptides, each composed of at least two protein species. Both VP-1 and VP-2 structural polypeptides were induced as heterogeneous phosphoproteins. The major VP-2 protein could be resolved in the form of a consistent pattern of three abundant (a to c), two intermediate (d and e), and one meager (f) neutral isoelectric focusing species or subtypes. This posttranslational modification precedes and is uncoupled from viral assembly, and all of the VP-2 subtypes are packaged into empty capsids at the induced stoichiometry. However, intracellular full virions harbored additional phosphorylated subtypes (g to l) and a subtle rearrangement in the whole VP-2 composition, while mature virions purified from lysed cultures lacked these subtypes, coordinately with the emergence of six neutral VP-3 subtypes. Thus, the virion coat undergoes a chemical transition entailed by genome encapsidation, in which phosphates seem to play a major role, triggering the preferential proteolytic cleavage of the more acidic VP-2 subtypes to VP-3. Parvoviruses, with small coding capacity, may regulate some morphogenetic steps, such as assembly, genome encapsidation, and maturation, by posttranslational modifications of their structural proteins.