Quantitative competitive polymerase chain reaction (QC-PCR) methods were used to quantify virion-associated human immunodeficiency virus type-1 (HIV-1) RNA in plasma from 66 patients with Centers for Disease Control stage I to IVC1 infection. HIV-1 RNA, ranging from 100 to nearly 22,000,000 copies per milliliter of plasma (corresponding to 50 to 11,000,000 virions per milliliter), was readily quantified in all subjects, was significantly associated with disease stage and CD4+ T cell counts, and decreased by as much as 235-fold with resolution of primary infection or institution of antiretroviral therapy. Plasma virus levels determined by QC-PCR correlated with, but exceeded by an average of 60,000-fold, virus titers measured by endpoint dilution culture. Quantitation of HIV-1 in plasma by QC-PCR may be useful in assessing the efficacy of antiretroviral agents, especially in early stage disease when conventional viral markers are often negative.

Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2′-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4+ target cells and mediated virus-induced, CD4-dependent “fusion from without” comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

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To define the contribution of CD8 + T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8 + T cell depletion using a rhesusized anti-CD8β monoclonal antibody (mAb) on barcoded SIVmac239 dynamics on stable ART and after ART cessation in Rhesus Macaques (RMs). Among the RMs with full CD8 + T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA + cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RM during ART. Upon ART cessation, both CD8 + T cell-depleted and control RMs rebounded in <12 days with no difference in the time to viral rebound, or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8 + T cell-depleted RMs showed a stable ~2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent anti-viral CD8 + T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

Though crystal structures for Env subunit fragments have been described, the overall structure of the Env spike and its distribution on virions has remained obscure. To address this issue, we have used cryoelectron microscopy (cryoEM) tomography to image unfixed and unstained HIV-1 and SIV virions in vitreous ice.Mutant (truncated TM tail) SIV virions show a surface fairly well saturated with 73 ± 25 rather uniformly distributed Env spikes, in line with our previous data. In striking contrast to SIV, yet consistent with our earlier results, cryoEM analysis of HIV-1 (MN) virions, which have a native non-mutated full-length tail, shows only 14 ± 7 Env/particle. An analysis of the Env spike distribution on HIV-1 virions shows a significantly higher proportion of spikes within clusters than predicted by chance alone. The clustering of Env spikes has implications for viral fusion and antibody-mediated neutralization mechanisms.To further investigate the surface features of these virions, we have produced a 3.2 nm resolution model of the SIV spike based on cryoEM tomogram 3D volume averages of over 6,000 spikes. The gp120 "head" of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. Our model shows that the TM "stalk" of each trimer spike is composed of three independent legs that project obliquely from the trimer head, tripod like. The model was fitted with available atomic structures of the unliganded gp120 core and the HIV-1 gp41 2F5 and 4E10 peptide epitopes and associated Fab fragments. The unusual open leg configuration of TM has implications for the design of future soluble Env trimer vaccine candidates. It also suggests a mechanistic explanation for the events leading up to membrane fusion wherein the three unassociated membrane-spanning domains (MSDs) and membrane-associated elements of the membrane proximal external domains (MPEDs) of a single spike can, collectively, encompass and disrupt the integrity of a relatively large area of the membrane, possibly by pulling upon and drawing the outer leaflet toward the target cell membrane upon triggering.