Targeted CPSF6 recoding reveals global fitness advantages of SPAD integration targeting and cell type-specific roles for capsid binding in HIV-1 DNA synthesis and nuclear import

As part of the CFIm complex, CPSF6 regulates cellular gene expression via alternative polyadenylation. CPSF6 binds primate lentiviral capsids and has been implicated in HIV-1 reverse transcription, nuclear import, and integration targeting. However, interpretations of the role(s) of CPSF6 during HIV-1 replication are complicated by the transcriptional changes that occur when the protein is depleted from cells. To disentangle the role of CPSF6 in HIV-1 replication from alternative polyadenylation, we selectively recoded its capsid-binding phenylalanine-glycine motif to alanine-alanine in CD4-positive Jurkat T cells, CEM12D7 T cells, and THP-1 myeloid/macrophage cells using CRISPR/Cas9-based homology-directed repair. While large variations in single-round HIV-1 infection were observed among isolated T cell clones, en masse comparisons of recoded T cells to cloned control T cells failed to reveal any significant differences in infection levels. Consistent with an overall fitness advantage provided by DNA integration in speckle-associated domains, which was ablated by recoding across cell types, HIV-1 replicated more robustly in control T cell populations. Single-round infection, in addition to spreading replication, was reduced in recoded myeloid/macrophage cells, which correlated with reductions in reverse transcription, and more robust blocks to HIV-1 nuclear import. Recoded monocytes/macrophages, however, did not reveal any evidence for enhanced type I interferon production or sensing of HIV-1 nucleic acids. Altogether, we define endogenously recoded cell lines to uncouple capsid binding from CPSF6’s role in RNA metabolism, which highlights a global fitness advantage of targeted DNA integration and cell type-specific roles for CPSF6 in HIV-1 reverse transcription, nuclear import, and virus replication proficiency. IMPORTANCE HIV-1 usurps cellular factors to effectively replicate and avoid innate immune sensing. HIV-1 capsid binds CPSF6, but the consequences of this virus-host interaction on HIV-1 biology are necessarily complicated by the cellular role of CPSF6 in pre-RNA processing. By endogenously recoding CPSF6 to selectively disrupt capsid binding while preserving its RNA-processing activity, we demonstrate in two different T cell lines that CPSF6-mediated targeting of HIV-1 DNA integration into nuclear speckle-associated domains correlates with optimal viral replication. Recoded THP-1 myeloid/macrophage cells revealed additional single-round and multi-round infection defects, which correlated with reductions in reverse transcription and HIV-1 nuclear import. These defects, however, did not correlate with enhanced type I interferon production or detection of HIV-1 nucleic acids by known innate immune sensors. Our work clarifies diverse roles for the capsid-CPSF6 interaction in HIV-1 infection of T lymphocytic versus myeloid cell lines and provides novel cell reagents for future research studies.