Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Abstract

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.

FIG. 1

Immunofluorescence analysis of BF cells persistently infected with wt SV5 that had been passaged 40 (p40) or 80 (p80) times. The cells were stained with either the anti-Pk or the anti-Pe MAbs, which recognize epitopes in either the N-terminus P/V common domain of the P and V proteins (anti-Pk) or the P-unique C-terminal domain (anti-Pe).

FIG. 2

Immunoblot analysis showing the relative levels of STAT1 in mock-infected, wt SV5-infected (48 h p.i.), or persistently infected BF cells that had been passaged 38 (p38), 66 (p66), or 111 (p111) times. Cells were (+; panel b) or were not (−; panel a) treated with IFN-α/β for 24 h prior to harvest.

FIG. 3

Comparison of the ability of the V proteins from wt SV5 and SV5 mci-2 to block IFN signaling in human and murine cells. Human 2fTGH (a) or murine BF (b) cells were transfected with 0.1 μg of pJATlacZ, 0.1 μg of the IFN-α/β-responsive plasmid, and either 0.3 μg of pEFlink2 (control plasmid), pEF.SV5-V/wt (that encodes the V protein of wt SV5), or pEF.SV5-V/SV5 mci-2 (that encodes the V protein of SV5 mci-2). At 40 h posttransfection, the culture medium was supplemented with IFN (+IFN) or left untreated (−IFN). After 4 h, the luciferase and β-galactosidase activities in cellular lysates were measured. The luciferase activity, expressed in relative light units, was normalized to the β-galactosidase activity.

FIG. 4

(a) The nucleotide sequence of the V/P gene of wt SV5 and rSV5-V/P N₁₀₀D (N > D) as confirmed by RT-PCR. (b) Comparison of the ability of wt SV5 and rSV5-V/P N₁₀₀D to block IFN signaling in human and murine cells. Human 2fTGH or murine BF cells were transfected with 0.1 μg of pJATlacZ, 0.1 μg of the IFN-α/β-responsive plasmid, and 0.3 μg of pEFlink2 (control plasmid). At 24 h posttransfection the cells were infected with either wt SV5 or rSV5-V/P N₁₀₀D. At 44 h posttransfection, the culture medium was supplemented with IFN (+IFN) or left untreated (−IFN). After 4 h, the luciferase and β-galactosidase activities in cellular lysates were measured. The luciferase activity, expressed in relative light units, was normalized to the β-galactosidase activity. (c) Immunoblot analysis showing the relative levels of STAT1 in total cell extracts made from BF cells that had been mock infected or infected with wt or rSV5-V/P N₁₀₀D Cells were harvested at 24, 48, or 72 h p.i. as indicated in subpanels a to c. In addition, extracts were made from cells that were harvested at 72 h p.i. but to which exogenous IFN-α/β was added at 24 h p.i. (subpanel d). The lower band (arrow) is an unidentified host cell protein.

FIG. 4

(a) The nucleotide sequence of the V/P gene of wt SV5 and rSV5-V/P N₁₀₀D (N > D) as confirmed by RT-PCR. (b) Comparison of the ability of wt SV5 and rSV5-V/P N₁₀₀D to block IFN signaling in human and murine cells. Human 2fTGH or murine BF cells were transfected with 0.1 μg of pJATlacZ, 0.1 μg of the IFN-α/β-responsive plasmid, and 0.3 μg of pEFlink2 (control plasmid). At 24 h posttransfection the cells were infected with either wt SV5 or rSV5-V/P N₁₀₀D. At 44 h posttransfection, the culture medium was supplemented with IFN (+IFN) or left untreated (−IFN). After 4 h, the luciferase and β-galactosidase activities in cellular lysates were measured. The luciferase activity, expressed in relative light units, was normalized to the β-galactosidase activity. (c) Immunoblot analysis showing the relative levels of STAT1 in total cell extracts made from BF cells that had been mock infected or infected with wt or rSV5-V/P N₁₀₀D Cells were harvested at 24, 48, or 72 h p.i. as indicated in subpanels a to c. In addition, extracts were made from cells that were harvested at 72 h p.i. but to which exogenous IFN-α/β was added at 24 h p.i. (subpanel d). The lower band (arrow) is an unidentified host cell protein.

FIG. 5

Analysis of ³⁵S-labeled polypeptides present in immune precipitates formed by the reaction of a pool of MAbs specific for the HN, NP, F, M, and P/V proteins with soluble antigen extracts made from BF cells that had been mock infected (a) or infected with wt SV5 or rSV5-V/P N₁₀₀D (N > D) (b to d). The cells were radioactively labeled from 20 to 24, 44 to 48, or 68 to 72 h prior to harvesting as indicated. (e) ³⁵S-labeled polypeptides present in the total cell extracts from rSV5-V/P N₁₀₀D-infected cells.

FIG. 6

BF cells were infected at a high MOI with wt SV5 or rSV5-V/P N₁₀₀D and passaged three times over a period of 2 weeks. At 24 h p.i. (a) and after the third passage (b), cells growing on coverslips in the tissue culture dishes were fixed and stained for immunofluorescence using an anti-NP MAb (NP-a).