Summary HIV-1 envelope glycoproteins (Envs) bind to CD4 receptor and CCR5/CXCR4 coreceptor and mediate viral entry ( Feng et al., 1996 ; Herschhorn et al., 2016 , 2017 ; Kwong et al., 1998 ). HIV-1 Envs are the sole target of neutralizing antibodies and a main focus of vaccine development ( Flemming et al., 2018 ). Here, we provide a step-by-step protocol to measure Env sensitivity to ligands, cold, and small molecules, as well as to study viral infectivity and to dissect parameters affecting HIV-1 Env function. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020) .

Highlights d HI.fate reports HIV-1 fate in target cells by using sensitive fluorescent proteins d Alternative fates of HIV-1 infection are linked to differential gene expression d Small molecules can modulate the fate of HIV-1 infection d Extra-cellular environment contributes to the fate of HIV-1 in vivo

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Many HIV strains downregulate the levels of CD4 receptor on the surface of infected cells to prevent superinfection. In contrast, the rare HIV-2 UC1 strain is noncytopathic and has no effect on CD4 expression in infected cells but still replicates as efficiently as more cytopathic strains in peripheral blood mononuclear cells (PBMCs). Here, we show that HIV-2 UC1 Env interactions with the CD4 receptor exhibit slow association kinetics, whereas the dissociation kinetics is within the range of cytopathic strains. Despite the resulting 10to 100-fold decrease in binding affinity, HIV-2 UC1 Envs exhibit long-lived activation state and efficient fusion activity. These observations suggest that HIV-2 UC1 Envs evolved to balance low affinity with an improved and readily triggerable molecular machinery to mediate entry. Resistance to cold exposure, similar to many primary HIV-1 isolates, and to sCD4 neutralization suggests that HIV-2 UC1 Envs preferentially sample a closed Env conformation. Our data provide insights into the mechanism of HIV entry.

Protection from human immunodeficiency virus (HIV) acquisition will likely require an effective vaccine that elicits antibodies against the HIV-1 envelope glycoproteins (Envs), which are the sole target of neutralizing antibodies and a main focus of vaccine development. Adjuvants have been widely used to augment the magnitude and longevity of the adaptive immune responses to immunizations with HIV-1 Envs as well as to guide the development of specific immune responses. Here we review the adjuvants that have been used in combination with HIV-1 Envs in several pre-clinical and human clinical trials in recent years. We summarize the interactions between the HIV-1 envelope glycoproteins and adjuvants, and highlight the routes of vaccine administration for various formulations. We then discuss the use of combinations of different adjuvants, the potential effect of adjuvants on the elicitation of antibodies enriched in somatic hypermutation and containing long complementarity-determining region 3 of the antibody heavy chain, and the elicitation of nonneutralizing antibodies.