HIV-1 capsid-targeting domain of cleavage and polyadenylation specificity factor 6

Abstract

The antiviral factor CPSF6-358 restricts human immunodeficiency virus type 1 (HIV-1) infection through an interaction with capsid (CA), preventing virus nuclear entry and integration. HIV-1 acquires resistance to CPSF6-358 through an N74D mutation of CA that impairs binding of the antiviral factor. Here we examined the determinants within CPSF6-358 that are necessary for CA-specific interaction. Residues 314 to 322 include amino acids that are essential for CPSF6-358 restriction of HIV-1. Fusion of CPSF6 residues 301 to 358 to rhesus TRIM5α is also sufficient to restrict wild-type but not N74D HIV-1. Restriction is lost if CPSF6 residues in the amino acid 314 to 322 interaction motif are mutated. Examination of the CA targeting motif in CPSF6-358 did not reveal evidence of positive selection. Given the sensitivity of different primate lentiviruses to CPSF6-358 and apparent conservation of this interaction, our data suggest that CPSF6-358-mediated targeting of HIV-1 could provide a broadly effective antiviral strategy.

Fig 1

CPSF6-358 residues 313 to 327 contribute to antiviral activity. (A) Summary of CPSF6-358 domain mapping by deletion mutagenesis. Briefly, HA-tagged CPSF6-358 was cloned into a pLPCX vector containing a puromycin gene as a selectable marker, and various CPSF6-358 deletion mutants were generated as described in the text. HeLa cells were transduced with CPSF6-358HA or CPSF6-358HA constructs carrying various deletions and then tested for their HIV-1 restriction by infecting cells with WT HIV-1 and N74D HIV-1. CMV, cytomegalovirus. Expression column: ++, high level; +, detectable; −, none. HIV Restriction column: +, strong; +/−, partial; −, none; na, not attempted. (B) (Top) WT or N74D HIV-RFP/VSV-G infection of HeLa cells expressing control vector, CPSF6-358, or various CPSF6-358 mutants as shown. After quantification of RFP-positive cells via FACS, percent infection relative to that for control vector-expressing cells was calculated. Error bars indicate standard deviations. (Bottom) Western blot analysis of introduced proteins expressed in stable cell lines.

Fig 2

Functional analysis of CPSF6-358 residues 313 to 327 by alanine scanning mutagenesis. (A) (Top) WT and N74D HIV-RFP/VSV-G infection of HeLa cells expressing CPSF6-358 mutants carrying single alanine mutations from residues 313 to 327. Percent infected cells was measured by FACS for RFP expression at 48 h postinfection. Error bars indicate standard deviations. (Bottom) Western blot analysis of stably transduced proteins. (B) Summary of alanine scanning mutagenesis. “a” represents single alanine mutants of CPSF6-358 with reduced antiviral activity. “A” represents alanine mutants of CPSF6-358 with minimal or no restriction of WT HIV-1.

Fig 3

Analysis of TRIM5/CPSF6 fusion proteins. (A) The B30.2/SPRY domain of rhTRIM5α was replaced by CPSF6(261–358), CPSF6(261–358)_ala, CPSF6(301–358), or CPSF6(301–358)_ala. (B) Lysates of HeLa cells stably expressing TRIM5 and TRIM5/CPSF6 fusion proteins were evaluated for relative protein expression levels by Western blot analysis. (C) HeLa cells expressing control protein and TRIM/CPSF6 fusion proteins were infected with WT HIV-RFP or N74D HIV-RFP. (D) HIV-1 DNA synthesis was measured by real-time qPCR. Genomic DNA extracted from cells infected WT HIV-RFP and N74D HIV-RFP was collected at various time points (0 h, 5 h, 11 h, and 24 h) and assayed by qPCR to detect RU5, second-strand transfer, and 2-LTR circle junction vDNA levels. Error bars indicate standard deviations.

Fig 3

Analysis of TRIM5/CPSF6 fusion proteins. (A) The B30.2/SPRY domain of rhTRIM5α was replaced by CPSF6(261–358), CPSF6(261–358)_ala, CPSF6(301–358), or CPSF6(301–358)_ala. (B) Lysates of HeLa cells stably expressing TRIM5 and TRIM5/CPSF6 fusion proteins were evaluated for relative protein expression levels by Western blot analysis. (C) HeLa cells expressing control protein and TRIM/CPSF6 fusion proteins were infected with WT HIV-RFP or N74D HIV-RFP. (D) HIV-1 DNA synthesis was measured by real-time qPCR. Genomic DNA extracted from cells infected WT HIV-RFP and N74D HIV-RFP was collected at various time points (0 h, 5 h, 11 h, and 24 h) and assayed by qPCR to detect RU5, second-strand transfer, and 2-LTR circle junction vDNA levels. Error bars indicate standard deviations.

Fig 4

A 58-amino-acid fragment of CPSF6 fused to AcGFP can block HIV-1 infection in a CA-dependent manner. (A) CPSF6-358, CPSF6(301–358), and CPSF6(301–358)_ala were fused to AcGFP. (B) Protein expression was detected by AcGFP antibody. (C) HeLa cells stably expressing AcGFP fused CPSF6 proteins were infected with WT HIV-RFP and N74D HIV-RFP. (D) Cells infected with WT HIV and N74D HIV were collected for genomic extraction at various time points (0 h, 5 h, 11 h, and 24 h). Real-time qPCR was performed to detect RU5, second-strand transfer, and 2-LTR circle junction vDNA levels. Error bars indicate standard deviations.