| Abstract: | 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. |
