Translation of turnip yellow mosaic virus RNA in vitro: a closed and an open coat protein cistron.

Sucrose gradient centrifugation of heat-denatured RNA of turnip yellow mosaic virus permitted the isolation of five RNA classes with molecular weights ranging from 2.0 to 0.25 X 10(6). The infectivity was shown to be confined to an RNA molecule of molecular weight 2.0 X 10(6). No significant increase in infectivity was obtained by combination of the latter RNA with the RNA classes of smaller size. Translation in vitro of the RNAs of different size classes in a wheat germ cell-free system revealed that the infectious RNA (molecular weight 2.0 X 10(6) does not promote the synthesis of the coat protein of turnip yellow mosaic virus. Efficient production of this coat protein was found exclusively when the smallest RNA class (molecular weight 250,000) was used as a messenger. It is concluded that RNA molecules of turnip yellow mosaic virus of molecular weight 2.0 X 10(6) contain a closed coat protein cistron and that RNA molecules of molecular weight about 2 to 3 X 10(5) with an open coat protein cistron can be isolated from the virions.     

Cauliflower mosaic virus coat protein is phosphorylated in vitro by a virion-associated protein kinase

A protein kinase has been found to be associated with particles of the plant virus cauliflower mosaic virus. This protein kinase can phosphorylate endogenous viral capsid proteins in vitro and exchange substrates with casein kinase type II. The activity is not affected by cAMP but is enhanced considerably by ADP. The cofactor is either Mn²⁺ or Mg²⁺, and the phosphate donor is either ATP or GTP. Serine and threonine residues are phosphorylated.     

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase-promoter complex

Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.     

An inhibitor of viral RNA replication is encoded by a plant resistance gene

The tomato Tm-1 gene confers resistance to tomato mosaic virus (ToMV). Here, we report that the extracts of Tm-1 tomato cells (GCR237) have properties that inhibit the in vitro RNA replication of WT ToMV more strongly than that of the Tm-1-resistance-breaking mutant of ToMV, LT1. We purified this inhibitory activity and identified a polypeptide of approximately 80 kDa (p80(GCR237)) using LC-tandem MS. The amino acid sequence of p80(GCR237) had no similarity to any characterized proteins. The p80(GCR237) gene cosegregated with Tm-1; transgenic expression of p80(GCR237) conferred resistance to ToMV within tomato plants; and the knockdown of p80(GCR237) sensitized Tm-1 tomato plants to ToMV, indicating that Tm-1 encodes p80(GCR237) itself. We further show that in vitro-synthesized Tm-1 (p80(GCR237)) protein binds to the replication proteins of WT ToMV and inhibits their function at a step before, but not after, the viral replication complex is formed on the membrane surfaces. Such binding was not observed for the replication proteins of LT1. These results suggest that Tm-1 (p80(GCR237)) inhibits the replication of WT ToMV RNA through binding to the replication proteins.     

Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.

The 3'- and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-G-U-A-G-A-A-A-C-A-A-G-G-...-C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenzing virus are formed by an internal deletion of the genomic RNA.     

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag–RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site.Full-length HIV-1 RNA has two critical functions in the viral replication cycle (Fig. S1): It serves as the viral genome in virions and as the template for the translation of group-specific antigen (Gag) and Gag-Pol polyproteins, which constitute the viral structural proteins and enzymes required for viral replication. HIV-1 RNA must travel to specific subcellular locations to serve its functions. Defects in RNA trafficking or mislocalization in the cytoplasm can affect not only the function of the viral RNA, making it unable to be packaged efficiently (1), but also the function of the proteins translated from the RNA, such as generating Gag with targeting and assembly defects (2–4). Although these studies highlight the importance of HIV-1 RNA trafficking in viral replication, the mechanism used to transport HIV-1 RNA in the cytoplasm remains undefined.The transport of cellular mRNA in the cytoplasm can be complex. Some mRNAs are transported in the cytoplasm predominantly by diffusion, whereas other mRNAs are actively transported along the cytoskeleton by motor proteins to specific locations in the cytoplasm (5–10). Currently, it is unclear whether HIV-1 RNA uses diffusion and/or motor transport to reach targeted locations. The cytoplasmic transport of HIV-1 RNA can be further complicated by the presence of viral protein components. During the early phase of HIV-1 expression, full-length RNA is transported in the cytoplasm without the influence of the viral structural protein or enzymes because Gag and Gag-Pol are encoded in the message. Attempts to visualize and follow HIV-1 RNA signals in the cytoplasm without Gag had not been successful (11). However, a study using total internal reflection fluorescence (TIRF) microscopy to observe the volumes near the glass–cell interface had shown that HIV-1 RNA not encoding Gag can be detected on plasma membrane (12). After translation of the unspliced RNA, Gag is present in the cytoplasm and can potentially interact with full-length RNA to affect its transport. Gag binds to full-length HIV-1 RNA to allow the specific packaging of the RNA genome into assembling particles. Although Gag–RNA complexes were detected in the cytoplasm in some studies (13), when and where the Gag–RNA interaction first occurs remain controversial (14, 15). Recent studies suggest that the Gag–RNA complex is transported in the cytoplasm via endosomal pathways for the assembly of nascent viruses (11, 16). Although some Gag–RNA complexes were observed to move in a directional manner, inhibition of the endosomal pathway does not reduce infectious virus production (11, 16, 17), which is not consistent with this suggestion. Compounded with the inability to visualize and follow cytoplasmic HIV-1 RNA signals in the absence of Gag (11), many basic questions regarding HIV-1 RNA trafficking remain unanswered.We previously described a system that can efficiently label and detect HIV-1 RNA in viral particles with single-RNA sensitivity (18). Briefly, HIV-1 genomes were engineered to contain binding sites for BglG, an Escherichia coli antitermination protein (19); because the binding sites are located in pol, only full-length viral RNAs contain these sequences. These full-length HIV-1 RNAs are functional; they can express encoded Gag protein and can be efficiently packaged into particles (18). When such HIV-1 RNAs are coexpressed with a modified BglG fused to a fluorescent protein, through binding of the fusion protein to viral RNA, fluorescent signals are efficiently detected in the viral particles. Furthermore, the detected fluorescent signals are specific, because such signals are not detected in viral particles containing HIV-1 RNA molecules lacking the BglG-binding sites (18).In this report, we sought to determine the major mechanism that transports HIV-1 RNA in the cytoplasm. By tagging HIV-1 RNA with a modified BglG protein fused to YFP (Bgl-YFP), we performed single-molecule tracking of HIV-1 RNA and found that most of the HIV-1 RNA moves dynamically in the cytoplasm in a nondirectional manner that is characteristic of diffusive movement. Furthermore, an intact cytoskeletal structure is not required for RNA movement. Although determining where Gag and RNA first interact is not our experimental goal, the presence of Gag may affect viral RNA transport. To address this issue, we studied HIV-1 RNA transport in cells that expressed sufficient levels of Gag for virus assembly and found that most RNAs are transported by diffusion. Finally, we tested the hypothesis that endocytosed viral particles contribute to the previously observed directional cotrafficking of Gag-RNA signals. Our results demonstrate that the directional transport of colocalized Gag-RNA signals has behavior similar to the behavior of the endocytosed particles or assembling complexes, indicating these signals are inbound endocytosed particles rather than outbound complexes for virus assembly. These results answer fundamental questions of HIV-1 biology and shed light on essential steps in viral replication.     

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

Retroviral Gag polyproteins coopt host factors to traffic from cytosolic ribosomes to the plasma membrane, where virions are released. Before membrane transport, the multidomain Gag protein of Rous sarcoma virus (RSV) undergoes importin-mediated nuclear import and CRM1-dependent nuclear export, an intrinsic step in the assembly pathway. Transient nuclear trafficking of Gag is required for efficient viral RNA (vRNA) encapsidation, suggesting that Gag:vRNA binding might occur in the nucleus. Here, we show that Gag is imported into the nucleus through direct interactions of the Gag NC domain with importin-α (imp-α) and the MA domain with importin-11 (imp-11). The vRNA packaging signal, known as ψ, inhibited imp-α binding to Gag, indicating that the NC domain does not bind to imp-α and vRNA simultaneously. Unexpectedly, vRNA binding also prevented the association of imp-11 with both the MA domain alone and with Gag, suggesting that the MA domain may bind to the vRNA genome. In contrast, direct binding of Gag to the nuclear export factor CRM1, via the CRM1-RanGTP heterodimer, was stimulated by ψRNA. These findings suggest a model whereby the genomic vRNA serves as a switch to regulate the ordered association of host import/export factors that mediate Gag nucleocytoplasmic trafficking for virion assembly. The Gag:vRNA interaction appears to serve multiple critical roles in assembly: specific selection of the vRNA genome for packaging, stimulating the formation of Gag dimers, and triggering export of viral ribonucleoprotein complexes from the nucleus.