Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components. The timing, location and mechanisms of HIV-1 capsid disassembly which is referred to as uncoating remains unclear. Direct labeling of HIV-1 capsids, by incorporating a few green fluorescent proteins (GFP) tagged capsid protein (CA) into virions allows to image the spatio-temporal loss of HIV-1 CA during virus infection. However, the localization and functions of a few virion incorporated eGFP-tagged CA proteins remain unclear, since <50% of virus packaged CA proteins participate to form the conical capsid shell that protects the HIV-1 genome. Here we developed several approaches to test the localization and function of eGFP-tagged CA proteins in virions. We found that eGFP-tagged CA proteins are excluded from the conical capsid shell and that a subset of these proteins is associated with the viral ribonucleoprotein complex (vRNPs), through direct interactions between CA and vRNP components. eGFP-tagged CA is retained in the nucleus by virtue of vRNP association and is unlikely to report on HIV-1 capsid disassembly. We also found that HIV-1 capsids become permeabilized and are remodeled during their transport into the nucleus. Our study provides new insights into the ability of CA to interact with vRNPs for its retention in the nucleus and highlights capsid remodeling as a preferred pathway for HIV-1 entry into the nucleus.