Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Abstract

Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4 ؉ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCECPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.T he HIV-1 capsid (CA) protein forms the protein core that envelopes viral enzymes and genetic material following fusion with the plasma membrane (1-3). Uncoating of the viral core is a fine-tuned process that regulates downstream events, including reverse transcription, nuclear entry, and integration targeting (4-9). It is likely that HIV-1 exploits multiple host factors that can directly interact with the viral CA protein for proper uncoating in virus-infected cells. These host factors likely in...

Capsid-Dependent Host Factors in HIV-1 Infection

“…Consistent with this model, NPCs harbor “transport”-independent functions that include chromatin organization and regulation of transcription [122]. Redirecting integration by heterologous LEDGF/p75 fusion proteins had little effect on viral gene expression [123, 124], but in vivo passage of an HIV-1 mutant with altered integration targeting selected for virus that restored CPSF6 binding and hence preferential integration into active euchromatin [75]. Thus, although the effects of integration targeting dictated by CPSF6 and other host factors are quantitatively subtle, they provide a critical fitness advantage for HIV-1 propagation under physiological conditions.…”

“…CPSF6 depletion, which tends to marginally increase virus infection [11, 65, 73] (Box 3), has been reported to reduce nuclear localization of viral DNA and CA [74] and integration [11]. Several CA amino acid substitutions in the CPSF6 binding pocket, including N74D and A77V, reduce the affinity of the virus-host interaction [65, 75]. The N74D mutant virus, though robust in T cell lines, is defective in macrophages due to inefficient reverse transcription [76] and/or innate immune activation [27] (Box 2).…”

“…The N74D mutant virus, though robust in T cell lines, is defective in macrophages due to inefficient reverse transcription [76] and/or innate immune activation [27] (Box 2). Importantly, A77V replicated similarly to the wild type (WT) virus in primary cells, yet lost out to the WT in head-to-head competition and reverted back to WT in the majority of inoculated humanized mice [75]. Thus, although CPSF6 is not an essential viral cofactor, HIV-1 prefers this interaction when push comes to shove.…”

“…This process is in part regulated by the capsid and CA-interacting proteins [54]. CA changes that specifically affect cofactor binding, as well as depletion of CA-binding host factors implicated in nuclear entry, such as, CypA, CPSF6, NUP358, and NUP153, alter integration site preference [8–11, 75, 111]. Thus, nuclear entry is functionally coupled with integration [8, 58].…”

“…HIV-1 or HIV-2 CA mutants with altered CypA binding elicit strong innate activation [26, 27, 91]. The CA mutant N74D, which is deficient for CPSF6 binding, induced type I IFN production in macrophages [27, 134], though one study did not reproduce this phenotype [75]. Viral DNA released from disassembled capsid cores is susceptible to degradation by the cytosolic exonuclease TREX1 to avoid innate sensing by DNA sensors [135].…”