Kenneth A. Matreyek

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved variants with substitutions in the spike receptor-binding domain (RBD) that affect its affinity for angiotensin-converting enzyme 2 (ACE2) receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites—a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single–amino acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently Asn 501 →Tyr (N501Y), cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change—for example, enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153(C)). NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153(C) under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C) and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C) mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C) for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C) expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C)-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

Lentiviruses likely infect nondividing cells by commandeering host nuclear transport factors to facilitate the passage of their preintegration complexes (PICs) through nuclear pore complexes (NPCs) within nuclear envelopes. Genome-wide small interfering RNA screens previously identified karyopherin β transportin-3 (TNPO3) and NPC component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). The knockdown of either protein significantly inhibited HIV-1 infectivity, while infection by the gammaretrovirus Moloney murine leukemia virus (MLV) was unaffected. Here, we establish that primate lentiviruses are particularly sensitive to NUP153 knockdown and investigate HIV-1-encoded elements that contribute to this dependency. Mutants lacking functional Vpr or the central DNA flap remained sensitive to NUP153 depletion, while MLV/HIV-1 chimera viruses carrying MLV matrix, capsid, or integrase became less sensitive when the latter two elements were substituted. Two capsid missense mutant viruses, N74D and P90A, were largely insensitive to NUP153 depletion, as was wild-type HIV-1 when cyclophilin A was depleted simultaneously or when infection was conducted in the presence of cyclosporine A. The codepletion of NUP153 and TNPO3 yielded synergistic effects that outweighed those calculated based on individual knockdowns, indicating potential interdependent roles for these factors during HIV-1 infection. Quantitative PCR revealed normal levels of late reverse transcripts, a moderate reduction of 2-long terminal repeat (2-LTR) circles, and a relatively large reduction in integrated proviruses upon NUP153 knockdown. These results suggest that capsid, likely by the qualities of its uncoating, determines whether HIV-1 requires cellular NUP153 for PIC nuclear import.