264 Evidence for a Cyclophilin-Dependent Host Factor in HIV-1 Infectivity
A. Saphire*, M. Bobardt, P. Gallay Scripps Res Inst, La Jolla, CA
Background: We previously demonstrated that cyclophilin A (CypA) plays a role in at least 2 distinct stages in HIV-1 replication: entry and post-entry.
Method: In order to examine the post-entry activity of CypA in HIV-1 infection, we employed an in trans Vpr-fusion system to introduce mutant forms of CypA into the virus under conditions that exclude the incorporation of endogenous CypA. Importantly, this Vpr-CypA chimera rescues the infectivity of viruses lacking a functional CypA packaging signal in the capsid (CA).
Results: Using this system, we found that a tryptophan CypA mutant (W121V), which has been shown to bind Gag but not CA, fails to rescue HIV-1 infectivity. This suggests that the CypA/CA interaction plays an important post-budding role, and occurs via a site distinct from the known CypA packaging signal on CA. In order to identify this domain, co-precipitation experiments were performed with recombinant CA mutants in regions that have glycine-proline (GP) motifs (GP156/157; GP206/207; GP223/224) reminiscent of that know n to bind CypA (GP89/90). Interestingly, one of these CA mutants binds CypA less efficiently, despite the presence of a wild-type GP motif at position 89/90. In the context of whole virus, this mutant is not infectious. Surprisingly, this mutant does incorporate CypA.
Conclusions: This suggests that although the GP 89/90 incorporation motif is functional, mutations in the post-incorporation target are not rescued by the presence of CypA. We are currently performing experiments to further identify residues in CypA re sp onsible for interaction with CA at this site in order to understand the post-budding activity of CypA on CA necessary for HIV-1 replication. An alternative explanation for our findings is that another host protein, working in conjunction with CypA, bi nds CA. In this scenario, this other protein binds CypA tryptophan 121, and CA via a GP motif. In order to explore this possibility, we are using Liquid Chromatographic coupled to Tandem Mass Spectrometry (LC/MS/MS) to compare the total protein content of wild-type viruses with viruses containing mutations either in glycine-proline CA motifs or in CypA (e.g., W121V). Furthermore, we are comparing wild-type HIV-1 with a mutant virus containing a small portion of the SIVmac CA region which replicates in the absence of CypA to determine if a host factor is incorporated via the SIVmac CA region and substitutes for CypA activity.
