The HIV life cycle requires ~ 24 hours for completion, under optimal in vitro conditions. Thus, irreversible entry into cells that have initiated an apoptotic program is a dead end for HIV, as these cells generally die within 24 hours. In vivo they may be cleared even more quickly by phagocytes recognizing surface markers such as everted PS. Virions that failed to irreversibly fuse with apoptotic cells would have a selective advantage over those that progressed beyond envelope binding to fusion. The potential importance of such discrimination is increased by the fact that HIV requires activated T cells for efficient entry, reverse transcription and integration. Since >90% of physiologically activated T cells are destined for activation induced cell death (AICD) over a period of ~ 5 days, there is a reasonable chance that HIV will encounter activated apoptotic CD4+ lymphocytes or blebs. This is particularly true during hyperacute disease in the gut, where there is massive CD4+ T cell infection in the setting of significant AICD. In the absence of specific anti-viral immune responses during the hyperacute phase, avoidance of apoptotic cell entry could be one dominant selective pressure. We hypothesize that this pressure has resulted in an ability of HIV to avoid irreversible fusion with apoptotic cells, and to remain infectious if bound. We further hypothesize that HIV+ DCs will not irreversibly transfer HIV to apoptotic cells via infectious synapses. We will also test two distinct but related hypotheses: apoptotic cells are defective in chemotaxis toward a source of shed HIV envelope, and defective in endocytosis of non-specifically bound virions.
Our specific aims are to 1) rigorously test these hypotheses using R5 envelope, virions, and HIV+ DCs, and 2) examine loss of receptor co-capping, and loss of LFA-1, as potential mechanisms whereby HIV may sense and avoid apoptotic potential host cells. Our long term objective is to exploit this feature of HIV to design prophylactic and treatment options that trick the virus into avoiding entry into healthy cells that have been made to mimic apoptotic cells in one or more aspects crucial to HIV infection. Our methods will use physiologically relevant signals to trigger AICD in CD4+ T cells, and expose them to HIV, or HIV+ DCs, at subsequent time points. Cells will be sorted for early apoptosis by fluorescent dye detected changes in mitochondrial membranes, and surface binding vs. irreversible entry of HIV will be detected by dual fluorescent virion entry, BlaM-vpr activity, and quantitative RT-PCR of intracellular genomic viral RNA, in conjunction with proteolytic stripping of HIV from the cell surface. Infectivity of HIV retained on the surface of apoptotic cells will be assessed by rescue cultures. This idea is completely novel, and is highly significant because it would provide insight into the evolution and structure of retroviral envelope, and would be the first demonstration of external probing by a pathogen of host cell fitness, prior to infection. Additional significance derives from the potential to exploit our findings for prevention and treatment, by pharmacologically conferring on key cellular components a transient apoptosis-mimicking phenotype.
HIV is a highly adaptable virus that evolves competitively within a single infected person to avoid elimination by host defenses or drugs, and to secure the best sites for replication within the body. In this Darwinian race among billions of individual virions, advantages in replication and survival rapidly lead to selection of the responsible traits, such as the ability of a virus to avoid entering a doomed potential host cell that has initiated a regulatory program of self-destruction, or a quiescent cell that cannot replicate virus efficiently.
We aim to demonstrate this avoidance mechanism, and, eventually, hope to pharmacologically mimic, transiently, key aspects of the cell that signal programmed cell death (or quiescence) to HIV, thereby tricking the virus into avoiding such cells.