Inactivation of Human Immunodeficiency Virus Type 1 Infectivity with Preservation of Conformational and Functional Integrity of Virion Surface Proteins

Abstract

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.

Membrane organization of virus and target cell plays a role in HIV entry

“…This method will be useful to verify previously obtained data on the behavior of single proteins in a more physiological and relevant context. Such an approach has already been successfully used with influenza viruses [89,90] and might be extended to inactivated HIV viruses that keep their binding and fusion properties [91,92]. Combined with the use of drugs to modulate lipid membrane composition these techniques will permit to understand the mechanisms involved in infection and more specifically the role played by lipids in this process.…”

Expression of PD-L1 and PD-L2 on human macrophages is up-regulated by HIV-1 and differentially modulated by IL-10

“…The mechanism(s) leading to the up-regulation of PD-L1 in HIV infection are unclear, although our results demonstrate a direct effect of the virus, as expression of PD-L1 within the macrophages infected with HIV-BaL and expressing p24 antigen was considerably higher than in the p24-negative population. Some up-regulation must occur during the early steps of the infection, before retrotranscription, as the AT-2-inactivated viruses used in our study are able to fuse with the membrane and enter the cells but cannot replicate [17]. Furthermore, the lack of infection and lack of up-regulation of PD-L1 in macrophages in the presence of NL4.3 HIV, a CXCR4-dependent virus, suggest that fusion or CCR5 use might be necessary for the up-regulation of PD-L1 and PD-L2.…”

“…Inactivated viruses were prepared as described previously [17]. Briefly, stocks of HIV-1 were purified and concentrated by centrifugation over a sucrose cushion and treated with AT-2, which covalently modifies the essential zinc fingers in the nucleocapsid protein of HIV-1.…”

“…After 5 days of differentiation, MDMs were stimulated for 48 h with noninfectious AT-2-treated HIV BaL or AT-2-treated HIV TZA (1.6 g/ml p24; lot P4047) [17]. As a negative control, MV, isolated from uninfected cell cul-tures, were used (lot P4063).…”

Macropinocytosis-Like HIV-1 Internalization in Macrophages Is CCR5 Dependent and Leads to Efficient but Delayed Degradation in Endosomal Compartments

“…This mild oxidizing reagent induces covalent modification of the free sulfhydryl groups of the cysteines of internal virion proteins, in particular, the nucleocapsid proteins. This inactivation method preserves the structural and functional integrity of Env glycoproteins on the virus surface and therefore has no impact on its interaction with cell surface receptors (33). Briefly, viral suspensions were incubated overnight at 4°C in the presence of 1 mM AT-2 and then purified by ultracentrifugation.…”