The HIV-1 Env Gp41 Cytoplasmic Tail: Key Functions During Cell-to-Cell Infection and Neutralization

Neutralizing antibody responses in HIV-1 infected individuals are directed against the HIV-1 envelope (Env) protein. The Env protein has evolved to avoid exposure of neutralizing epitopes, and maintain its ability to mediate viral entry. Direct T cell-to-T cell HIV-1 infection through virological synapses (VS) has been increasingly recognized as a major mode of infection during in vitro co-culture of infected and uninfected T cells and has the potential to make significant contributions to overall infection in vivo, warranting further investigation of this mode of infection. Here, we assessed the neutralization of cell-to-cell infection by polyclonal patient sera from two HIV-1 positive individuals, as well as a panel of broadly neutralizing monoclonal antibodies (mAbs). Neutralization of cell-to-cell infection required higher concentrations of patient sera and a 7 to 70-fold higher 50% inhibitory concentration (IC50) of the anti-Env mAbs compared to cell-free infection. Recognizing the influence of the gp41 cytoplasmic tail (CT) on Env conformation and function during cell-free infection, we next carried out neutralization assays on an HIV-1 mutant with a full truncation of the gp41 CT, ?CT144. Despite higher levels of cell-surface Env expression, CT truncation specifically enhanced neutralization in our cell-to-cell assays, with little or no effect on the IC50 of cell-free infection. Although ?CT144 can replicate in vitro in specific permissive cell types, systematic truncation does not always produce infectious HIV-1 particles. Because of the key role that the Env CT plays in Env packaging, viral fusion and subsequent cell-free infectivity, we were interested in determining if these functions were conserved during cell-to-cell transmission through the VS. We identified viral mutants that were severely defective in either cell-free or cell-to-cell infection, providing further evidence that these two modes of infection are distinct. Taken together, our results indicate that there are (i) conformational differences between virion-associated and cell-associated Env that influence neutralization sensitivity, and (ii) mechanistic differences that distinguish how the Env CT participates in cell-free or cell-to-cell infection.