The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.

The assembly and release of retroviruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retrovirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retrovirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer monkey virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1-and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retrovirus release from host cells and in cell-cell transmission events. IMPORTANCEViruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retrovirus, Mason-Pfizer monkey virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroviruses.

HIV-1 assembly and release occurs at the plasma membrane of human T lymphocytes and model epithelial cell lines, whereas in macrophages intracellular sites of virus assembly or accumulation predominate. The origin of the intracellular virus-containing compartment (VCC) has been controversial. This compartment is enriched in markers of the multivesicular body, and has been described as a modified endosomal compartment. Several studies of this compartment have revealed the presence of small channels connecting to the plasma membrane, suggesting that instead of an endosomal origin the compartment is a modified plasma membrane compartment. If the compartment is accessible to the external environment, this would have important implications for antiviral immune responses and antiviral therapy. We performed a series of experiments designed to determine if the VCC in macrophages was open to the external environment and accessible to antibodies and small molecules. The majority of VCCs were found to be inaccessible to exogenously-applied antibodies to tetraspanins in the absence of membrane permeabilization, while tetraspanin staining was readily observed following membrane permeabilization. Cationized ferritin was utilized to stain the plasma membrane, and revealed that the majority of virus-containing compartments were inaccessible to ferritin. Low molecular weight dextrans could access only a very small percentage of VCCs, and these tended to be more peripheral compartments. We conclude that the VCCs in monocyte-derived human macrophages are heterogeneous, but the majority of VCCs are closed to the external environment.