Kyusik Kim, Ann Dauphin, Sevnur Komurlu, Sean M. McCauley, Leonid A. Yurkovetskiy, Claudia Carbone, William E. Diehl, Caterina Strambio-De-Castillia, Edward M. Campbell, and Jeremy Luban. Cyclophilin A protects HIV-1 from restriction by human TRIM5α. Nature Microbiology is online today and the full PDF can be accessed here.

In 1993, in one of the first two-hybrid screens of a mammalian cDNA library for encoded proteins that interact with a protein of interest, undergraduate student Karen Bossolt discovered that the HIV-1 Gag polyprotein, as well as the mature capsid protein (CA) embedded within it, bind to the cellular protein cyclophilin A (CypA). In 2001, graduate student Douglas Braaten formally demonstrated the importance of this interaction for HIV-1 replication in what was perhaps the first targeted gene disruption experiment by homologous recombination in somatic cells. Over the years, attempts to determine the mechanism of action of cyclophilin A have nonetheless taken many unexpected turns.

Undergraduate student Ettaly Franke found that Cyclophilin A was incorporated into HIV-1 virions at a molar ratio ~1:5 with respect to CA. This suggested that cyclophilin A played a role during virion assembly or within the virion itself. This idea was supported by the observation that infectivity decreased when virions were produced in the presence of cyclosporine, a competitive inhibitor of the Gag-CypA or CA-CypA interaction. Ultimately, graduate student Elena Sokolskaja demonstrated that the effects of cyclosporine during virion assembly were independent of the CA-CypA interaction and could be attributed to disruption of Env glycoprotein incorporation into particles. Elena also showed that HIV-1 CA interaction with TARGET CELL cyclophilin A was important for HIV-1 infectivity. Still today, it remains an open question whether cyclophilin A plays any role during HIV-1 virion assembly, or as a virion component.

Several observations suggested that cyclophilin A protected the incoming HIV-1 CA core from a dominant-acting, antiviral factor within human target cells. Among other properties, the putative antiviral factor was cell type-specific, species-specific, saturated by large amounts of susceptible virion cores, and disrupted by arsenic. Then, in 2004, two screens identified TRIM5 as a species-specific, capsid-specific, restriction factor. The first screen, which used a macaque cDNA expression library, was conducted by graduate student Matt Stremlau in the Sodroski lab. He showed that the macaque TRIM5α orthologue restricted HIV-1; he was unable to detect this anti-HIV-1 activity with the human TRIM5α orthologue. The second screen was conducted by graduate student David Sayah who screened an owl monkey cDNA library. David discovered that the potent anti-HIV-1 restriction activity in this species was also due to the TRIM5 gene, but with a twist: a CypA cDNA had retrotransposed into the owl monkey TRIM5 locus to create a TRIM5-CypA fusion gene that is among the most potent anti-HIV-1 restriction factors. Amazingly, there is now evidence that CypA cDNA has retrotransposed into the TRIM5 locus at least 9 separate times over the course of phylogeny to create fusion genes. Presumably these TRIM5-CypA fusion genes were selected for by challenges with lentiviral ancestors of HIV-1.

The above experiments suggested that, in human cells, CypA might protect HIV-1 from restriction by TRIM5α. Experiments conducted by Elena in the early days of RNAi indicated that the effects of CypA and TRIM5α were independent of each other when HIV-1 infection was measured on tumor cell lines. Since then, working on a range of different projects, a line of graduate students that includes Thomas Pertel, Nadia Rahm, Martha Neagu, Alberto De Iaco, and Sean McCauley, have improved the efficiency of lentiviral vector-based reverse genetic tools for gene disruption, and for gene delivery, in primary human blood cells. Most recently, graduate students Kyusik Kim and Sean McCauley developed what are the most efficient lentiviral vectors yet.

While studying unrelated host factors that he hypothesized to regulate HIV-1 infection, Kyusik Kim fortuitously disrupted CypA in primary human dendritic cells; CypA had been intended as a negative control since he expected it to have little effect in that specific experiment. To his surprise, CypA knockdown had a qualitatively bigger effect on HIV-1 replication in primary human dendritic cells than it had had in the tumor cell lines that had been used previously. Prior to this, our lentiviral vectors were not efficient enough to disrupt CypA to this extent in dendritic cells. Kyusik found the same impressive CypA knockdown phenotype in primary human macrophages and CD4+ T cells. As described in his new paper, Kyusik shows pretty conclusively that HIV-1 replication is very dependent on CypA in primary cells. But then, most surprising, and perhaps most important of all, Kyusik found that CypA protects HIV-1 from the potent restriction activity of endogenous human TRIM5α in these cells. Until now, the human orthologue of TRIM5α was thought to have little effect on HIV-1 replication.

Congratulations to Kyusik and his collaborators, Ann, Sevnur, Sean, Leonid, Claudia, Ted, Caterina, and Ed!!!!

Kyusik Kim