Correction of a carboxyl terminal simian immunodeficiency virus Nef frameshift mutation restores virus replication in macaques

Abstract

Previous studies demonstrated that the nef gene is a critical determinant of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. In the present study, we evaluated the effect of a spontaneous frameshift mutation in the C-terminus of the nef gene of the minimally pathogenic SIVsmH4i clone. This clone exhibited a single nucleotide deletion in the nef gene relative to pathogenic SIV clones that resulted in a frameshift and addition of 46 amino acids to the C-terminus of Nef. We generated a corrected version of this clone, SIVsmH4i Nef+ that restored Nef protein expression. Inoculation of macaques with SIVsmH4i resulted in delayed and low levels of peak viremia. This contrasted with improved kinetics and robust peak viremia in macaques inoculated with the corrected version. Despite the restoration of in vivo replication ability, neither clone resulted in memory CD4+ T cell loss or disease in a period of two years.

Fig. 1

Comparison of nucleotide (A) and amino acid (B) sequences of the C-terminus of Nef of the original SIVsmH4i, the corrected version and sequences found at various time points post-infection in macaques H729. The nucleotide sequence of the original clone at the top is aligned with nucleotide substitutions below and identical nucleotides indicated by a dash (-). Gaps are indicated by a dot (.). The amino acid sequence of the C-terminus of Nef is shown in single amino acid code using the same symbols.

Fig. 2

Western blot analysis of the Nef protein expression in SIVsmH4i and SIVsmH4I Nef+-transfected 293T cells. A. Western blotting with RhE544 anti-SIV serum, demonstrates a 32 kDa protein consistent with the size of Nef in both SIVsmH4i Nef+ and SIVmac239 infected cells expressing Nef protein. A comparable but larger molecular weight species cannot be identified in SIVsmH4i-transfected cells. B. SIVsmH4i, SIVsmH4in, and SIVmac239 infected 293T cell lysate western blotting with anti-SIVmac251 Nef monoclonal antibody, which only recognizes SIVmac239 Nef protein, confirms that the 32 kDa protein expressed in panel A is indeed Nef.

Fig. 3

Comparison of replication of SIVsmH4i and SIVsmH4i Nef+ in CEMx174 cells and PBMCs in vitro. A. SIVsmH4i Nef+ virus replicated more efficiently than the parental SIVsmH4i virus in CEMx174 cells (P<0.05). B. SIVsmH4i Nef+ virus replicated more efficiently than the parental SIVsmH4i virus in rhesus PBMC cells (P<0.05).

Fig. 4

Comparison of the ability of SIVsmH4i and SIVsmH4iNef+ to down-regulate MHC-I and CD4 on cell surface following infection of M7-Luc cells. M7-Luc cells were infected with SIVsmH4i, SIVsmH4iNef+ or SIVsmE543-3 in triplicate and tested at 68 h post-infection. To measure the level of MHC-I and CD4, cells were stained with anti-human CD4-APC, 7-AAD and HLA-A2-PE. Approximately 30,000 live infected cells (as determined by EGFP positive and 7-AAD negative gating) were counted by flow cytometry and FACS histograms of cell distribution for representative samples are shown in the upper panels. The mock-infected M7-Luc cells were used to set the MHC-I and CD4 positive gates and infected M7-Luc cells stained with isotype control antibodies were used to set the negative gates. The lower panels show the percentage of cells which down-regulated MHC-I or CD4 molecules as indicated by HLA-A2 low or CD4 low expression. The brackets on the graphs represent 95% CI and numbers represent the P value for the unpaired t-test comparing means of indicated triplicates of cells infected with different SIVs.

Fig. 5

Comparison of plasma viremia and CD4 T cell counts in peripheral blood of SIVsmH4i (left panels) and SIVsmH4i Nef+ (right panels). A. Plasma viral RNA levels are shown sequentially throughout infection of each of the macaques. B. Absolute CD4+ T cell numbers in peripheral blood are shown sequentially for all macaques. C. The percent of preinoculation CD4+ T cells remaining at various time points after infection is shown.

Fig. 6

Sequential comparison of mean plasma viremia (A), absolute CD4+ T cell counts (B) and percent CD4 cell remaining (C) in peripheral blood of SIVsmH4i (unfilled squares) and SIVsmH4i Nef+ (black circles) inoculated macaques. Mean plasma viremia (A) and CD4+ T cell counts (B) are also compared to a cohort of six SIVsmE543-3-infected rhesus macaques (grey symbols), a fully pathogenic infection.