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

ABSTRACT 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.