HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV MN . Interestingly, plasma mvNef levels in HIV þ patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIVinfected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.

In the era of combined anti-retroviral therapy (CART) many of the complications due to HIV-1 infection have diminished. One exception is HIV Associated Neurocognitive Disorders (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report we show that Nef protein and nef mRNA are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of Aβ and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

20Human Immunodeficiency Virus (HIV) accessory protein Negative factor (Nef) is detected in the 21 plasma of HIV+ individuals associated with exosomes. The role of Nef+ exosomes (exNef) in HIV 22 pathogenesis is unknown. We perform a retrospective longitudinal analysis to determine 23 correlative clinical associations of exNef plasma levels in ARV-treated HIV+ patients with or 24 without immune recovery. exNef concentration in a subset of AIDS Clinical Trial Group (ACTG) 25 384 participants with successful virological suppression and with either high (∆ >100 CD4 cell 26 recovery/High Immunological Responders (High-IR) or low (∆ ≤100 CD4 cell recovery/ Low 27 Immunologic Responders (Low-IR) immunologic recovery was measured and compared for 28 study weeks 48, 96, and 144. CD4 recovery showed a negative correlation with exNef at study 29 week 144 (r = -0.3573, *p=.0366). Plasma exNef concentration in high IRs negatively correlated 30 with naïve CD4 count and recovery (r = -0.3249, *p = 0. 0348 (High-IR); r =0.2981, *p= #0.0513 (Low-31 IR)). However, recovery of CD4 memory cells positively correlated with exNef (r =.4534, 32 *p=.0358) in Low-IRs but not in High-IRs. Regimen A (Didanosine, Stavudine, Efavirenz) lowered 33 exNef levels in IRs by 2-fold compared to other regimens. Nef+ exosomes persist in ART-treated 34 HIV+ individuals despite undetectable viral loads, negatively correlates with naive and memory 35 CD4 T cell restoration and may be associated with reduced immunological recovery. Taken 36together, these data suggest that exNef may represent a novel mechanism utilized by HIV to 37 promote immune dysregulation. 38 39 41The prognosis for patients infected with HIV has improved significantly with the advent of 42 combination antiretroviral therapy (cART) which can lead to suppression of plasma viremia 43 usually associated with marked improvements in CD4+ T cell counts [1, 2]. However, a subset of 44 patients do not experience a robust immune recovery despite viral suppression. While host 45 factors such as age and baseline CD4 + T-cells with a naïve phenotype have been observed to be 46 negatively associated with the magnitude of CD4 + T-cell count improvement [1,[3][4][5][6], virological 47 factors that contribute to these immunologic outcomes are less well defined. 48

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