CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release.

Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation.

HIV-1 Tat protein contributes to HIV-neuropathogenesis in several ways including its ability to be taken up by uninfected bystander CNS cells and to activate inflammatory host genes causing synaptic injury. Here, we report that in the globally dominant HIV-1 clade C, Tat displays a naturally occurring polymorphism, R57S, in its basic domain, which mediates cellular uptake. We examined the effect of this polymorphism on Tat uptake and its consequences for cellular gene transactivation. In decapeptides corresponding to the basic domain, a R57S substitution caused up to a 70% reduction in uptake. We also used a transcellular Tat transactivation assay, where we expressed Tat proteins of HIV-1 clade B (Tat-B) or C (Tat-C) or their position 57 variants in HeLa cells. We quantified the secreted Tat proteins and measured their uptake by TZM-bl cells, which provide readout via an HIV-1 Tat-responsive luciferase gene. Transactivation by Tat-B was significantly reduced by R57S substitution, while that of Tat-C was enhanced by the reciprocal S57R substitution. Finally, we exposed microglia to Tat variants and found that R57 is required for maximal neuroinflammation. The R57S substitution dampened this response. Thus, genetic variations can modulate the ability of HIV-1 Tat to systemically disseminate neuroinflammation.

Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup.

HIV-1 Tat protein is secreted from infected cells and is endocytosed by uninfected bystander cells. Subsequently, Tat is translocated to the nucleus and binds to promoters of host cell genes, increasing the production of inflammatory host cytokines and chemokines. This inflammatory activation of uninfected cells by HIV-1 Tat protein contributes to the overall inflammatory burden in the central nervous system (CNS) that leads to the development of HIV-associated neurocognitive disorders (HAND). Here we describe methods to evaluate the uptake and transcriptional impact of HIV-1 Tat on uninfected cells by using a trans-cellular transactivation system. Cell lines transiently transfected with Tat expression constructs secrete Tat into the culture medium. Trans-cellular uptake and transactivation caused by secreted Tat can be measured by co-culturing LTR-responsive reporter cells with Tat-transfected cells. Such Tat-producer cells can also be co-cultured with immune cell lines, such as monocytic THP-1 cells or lymphocytic Jurkat T-cells, to evaluate transcriptional changes elicited by Tat taken up by the uninfected cells.

Viral and cellular factors underlying neuropathogenesis in HIV associated neurocognitive disorders (HAND).

As the HIV-1 epidemic enters its fourth decade, HIV-1 associated neurological disorders (HAND) continue to be a major concern in the infected population, despite the widespread use of anti-retroviral therapy. Advancing age and increased life expectancy of the HIV-1 infected population have been shown to increase the risk of cognitive dysfunction. Over the past 10 years, there has been a significant progress in our understanding of the mechanisms and the risk factors involved in the development of HAND. Key events that lead up to neuronal damage in HIV-1 infected individuals can be categorized based on the interaction of HIV-1 with the various cell types, including but not limited to macrophages, brain endothelial cells, microglia, astrocytes and the neurons. This review attempts to decipher these interactions, beginning with HIV-1 infection of macrophages and ultimately resulting in the release of neurotoxic viral and host products. These include: interaction with endothelial cells, resulting in the impairment of the blood brain barrier; interaction with the astrocytes, leading to metabolic and neurotransmitter imbalance; interactions with resident immune cells in the brain, leading to release of toxic cytokines and chemokines. We also review the mechanisms underlying neuronal damage caused by the factors mentioned above. We have attempted to bring together recent findings in these areas to help appreciate the viral and host factors that bring about neurological dysfunction. In addition, we review host factors and viral genotypic differences that affect phenotypic pathological outcomes, as well as recent advances in treatment options to specifically address the neurotoxic mechanisms in play.