Capsid labelled HIV to investigate the role of capsid during nuclear import and integration

Abstract

The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating eGFP-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP containing complexes. Low numbers of CA-eGFP molecules are located inside the viral core and imported in the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retain both labelled proteins after nuclear entry implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP containing complexes are detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.