CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes

Abstract

The ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3 (>10-fold) and HIV-1LAI (2- to 5-fold) in multiple human cell lines and primary CD4+ T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3 but not HIV-1LAI. The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAI selected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAI but not HIV-1NL4-3. The rescue of N57A by G94D in HIV-1LAI is abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3 and HIV-1LAI CA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types. IMPORTANCE The specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

A Novel Phenotype Links HIV-1 Capsid Stability to cGAS-Mediated DNA Sensing

“…8C). WT LAI virus had a steady decrease in RNA staining after the initial time point, similar to previous results (55,72,73). In contrast, higher numbers of RNA-positive particles were observed at 60 and 90 min postinfection for the CH040 virus than for the LAI virus, consistent with delayed core permeabilization.…”

“…Capsid permeabilization assay. 293T cells (ATCC) were transfected with (i) pBru3ori-ΔEnv-luc2, (ii) pVpr-mRuby3-IN (72), and (iii) pCMV-VSV-G plasmids using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) in the presence of 5-ethynyl uridine (EU), as previously described (55,73). After 48 h, the cell supernatant was collected and filtered through a 0.45-m-pore-size polyethersulfone syringe filter (Millipore).…”

“…To further examine whether the stable cores were intact or partially opened, we utilized an alternative imaging method, which takes advantage of the dependence of viral RNA staining on the opening of the viral capsid (55,72,73). Previous work showed that the 5-ethynyl uridine (EU) incorporated into HIV-1 RNA during virus production can be stained in cells after virus infection, with a decrease in RNA detection over time occurring concurrently with uncoating and reverse transcription (55).…”

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor

“…The CA-N57A mutation is deleterious for HIV-1 infection 54 and for binding of CA hexamers to FG motifs of Nup153 and CPSF6 (refs. 36 , 40 ; Extended Data Fig.…”

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

“…Exchanges were attempted randomly between successive replicas; trial attempts for all replicas were equally probable. The following Metropolis Monte Carlo exchange criterion42 wasemployed ℎ = min (1, − [ ( )− � �]+[ � �− ( )] )Where T=310 K, is the Boltzmann constant, and denote the 3D conformations for two replicas i and j, and and represent the potential energies derived from the restrained Hamiltonian evaluated at the indicated conformation.…”