The nuclear pore (NPC) and its components (nucleoporins) have long been implicated as host factors involved in HIV-1 infection. Nucleoporins facilitate the entry of the preintegration complex (PIC) into the nucleus but when and where these nucleoporins are recruited to facilitate translocation is still unclear. While assaying CRM1 dependent unsplice HIV-1 RNA genome export from the nucleus to the cytoplasm, we recently found that the RNAs do not export in a canonical manner through an NPC. Rather, frequently docking and interacting with the NPCs, but almost never translocating across them, the HIV-1 RNA 'absorbs' nucleoporin (Nup) components resulting in HIV-1 RNA foci that colocalize with the Nups, and lower Nup density per NPC. The NPCs, concomitantly to or as a result of the loss of Nups cluster along the nuclear envelope (NE) leaving prominent gaps between pores. Within these gaps, bulges and herniations appear which constitute the points of en masse HIV-1 RNA exodus into the cytoplasm. The hijacked Nups colocalize with the viral genomes into the cytoplasm, continue with them to the cell membrane and are copackaged with the genomes into the newly forming viral particles that release into the intracellular space. This finding has been verified with different cell lines, different HIV-1 mode genomes and we show that the process is reliant, at least, on fully functional Rev and HIV-1 genome RNA. Within this proposal we probe the mechanism of this finding, comparing it to host cell CRM1 mediated mRNA export and the recently described 'nuclear budding' process. We verify its occurrence in primary T and macrophage cells and elucidate which additional nucleoporins (and other host proteins) are involved in the process. We next address how the Rev-HIV-1 RNA interaction (through the Rev Response Element, RRE) affects this process, by concentrating on the Rev protein and its oligomerization host factor, DDX1. Using a combination of Rev mutants and beyond state of the art microscopy (termed SMRT - single molecule real time microscopy), and our recently developed image analysis tools, we provide a spatial and temporal landscape to the Rev:Rev and Rev:RRE interactions together with host factors including CRM1, DDX1 and nuclear pore components. SMRT microscopy allows for the simultaneous detection of individual HIV-1 genomes, host and viral proteins in real time within the living cell at low excitation power, =100 images/second speed and ~20 nm precision. The discovery of a potentially novel RNA export mechanism has far reaching implications both for fundamental cell biology and the HIV-1 field itself. Probing these binding cascades as they occur in the living cell also provides an exciting possibility to correlate elegant biochemical and in vitro knowledge with where and when exactly they occur in the cell.
We have discovered a novel pathway that the HIV-1 virus uses to export its viral genetic material from the nucleus to the cytoplasm in an infected cell. We will study this further, potentially elucidating therapeutic targets that may finally eradicate thi disease.
