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Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication - PubMed

Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication

Machiko Nishio et al. J Virol. 2005 Jul.

Abstract

Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.

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Figures

FIG. 1.
FIG. 1.

V-specific regions of paramyxovirus V proteins. The V-specific regions of six paramyxoviruses and a chimeric virus, PIV2/SeV-H178W/E182W, were aligned. The amino acids are numbered from the amino terminus of each V protein. The asterisks indicate conserved cysteines; squares indicate conserved tryptophans.

FIG. 2.
FIG. 2.

V protein complex formation with STATs. (A and B) BSR T7/5 cells were transfected with either pFlag-STAT1 or pFlag-STAT2 plus pTM1 carrying one of the V genes indicated in panel A (lanes 2 to 5, panel B) or an empty pTM1 (lanes 1). Whole-cell extracts were prepared at 48 h posttransfection, and samples containing equal amounts of total protein were assayed by Western blotting for their levels of Flag-STAT1 or Flag-STAT2 (anti-Flag) and V proteins (anti-V). Other samples were first immunoprecipitated (IP) with Flag affinity gel (Sigma), and the selected materials were then assayed by Western blotting for their levels of V proteins. (C and D) The same experiment as above, except that tryptophan mutants are examined, and only the results of STAT2 complex formation are shown. The asterisks on the right indicate the immunoglobulin light chain.

FIG. 3.
FIG. 3.

Effects of V proteins on IFN-α-stimulated gene activation. This experiment was carried out as described in Materials and Methods. Briefly, 2fTGH cells were transfected with a reporter plasmid [pISRE-(f)-luc] and a V expression vector (pEBS-V) or relevant control (see text), along with pTK-(r)-luc as an internal reference for transfection efficiency. After incubation for 24 h, the cells were treated with or without IFN-α2/α1 (1,000 IU/ml) for 14 h and then lysed. The levels of both firefly (f) and Renilla (r) luciferase activities were determined. Data represent the mean values of the normalized firefly luciferase activities from triplicate samples. The V proteins in expression vectors used in this experiment are mumps virus (MuV) V, mutant MuV V (C189/193A, C214/217A, W184H/W188E, and F139S), hPIV2 V, and mutant hPIV2 V (W178H/W182E, C193/197A, C209/211/214A, C218/221A, and F143S) proteins. Green fluorescent protein alone and Sendai virus C protein (SeV C) were used as negative and positive controls, respectively.

FIG. 4.
FIG. 4.

STAT1 and STAT2 levels in HeLa cells constitutively expressing V proteins. Samples of cytoplasmic extracts containing equal amounts of total protein of HeLa cells constitutively expressing various V proteins (listed above each lane) or nonexpressing HeLa cells (lanes 1) were assayed by Western blotting for their levels of endogenous STAT1 and STAT2, as well as that of the various V proteins, using anti-P/V, anti-STAT1, and anti-STAT2, as indicated to the left of each panel. The asterisk on the right indicates a cross-reacting host band. These results, and those of other mutants (data not shown), are summarized in Table 1.

FIG. 5.
FIG. 5.

Growth kinetics of rPIV2s carrying V protein mutations in tissue culture. Monolayers of Vero, HeLa, and PMK cells were infected with the various rPIV2s (A) at an MOI of ca. 0.01. Samples of the supernatants were harvested at various times postinfection, and their titers (PFU/ml) were determined by plaque formation on Vero monolayers (Materials and Methods). The symbols below the sequences indicate the positions of the mutations. (B). The three groups of virus mutants are listed (for details, see text).

FIG. 6.
FIG. 6.

Generation of a recombinant hPIV2 which completely lacks expression of V protein (rPIV2/P-edit). (A) The expressed ORFs of the wt P and V mRNAs and the P-edit P mRNA are shown as boxes. The positive-sense nucleotide sequences around the editing site of each mRNA are shown above; the two pseudotemplated G's added during P mRNA synthesis are underlined. The encoded protein sequence is shown in single-letter code. Nucleotides in lowercase letters show the six silent mutations used to inactivate the mRNA editing signal (AAGAGGGGGGG). The expected patterns of P gene expression are indicated. (B) Samples of cytoplasmic extracts of wt rPIV2 (lane 1)- and rPIV2/P-edit (lane 2)-infected Vero cells (72 hpi) were examined by Western blotting with anti-P/V MAb.

FIG. 7.
FIG. 7.

STAT2 levels of HeLa cells infected with rPIV2s. HeLa cell monolayers were infected with the various rPIV2s listed above lanes 2 to 11, at an MOI of 3, or mock infected (lane 1). Cytoplasmic extracts were prepared at 48 hpi, and samples containing equal amounts of total protein were examined by Western blotting for their levels of P and V proteins and endogenous STAT1 and STAT2. Actin levels were also examined as a loading control. The asterisk beside the STAT2 blot indicates an unknown host band that also may serve as a loading control.

FIG. 8.
FIG. 8.

STAT2 levels of Vero, BHK, L929, and MDCK cells infected with rPIV2s. Vero cell (monkey) (A), BHK cell (hamster) (B), L929 cell (murine) (C), and MDCK cell (canine) (E) monolayers were infected with the various rPIV2s listed above the lanes at an MOI of 3 or mock infected (lanes 1). Cytoplasmic extracts were prepared at 48 and 72 hpi, and samples containing equal amounts of total protein were examined by Western blotting for their levels of P and V proteins and endogenous STAT1 and STAT2. Actin levels were also examined as a loading control. Panel D shows Western blots of two independent L929 cell lines constitutively expressing hPIV2 V protein (lanes 2 and 3) and their progenitor cells (lane 1).

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References

    1. Andrejeva, J., E. Poole, D. F. Young, S. Goodbourn, and R. E. Randall. 2002. The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5. J. Virol. 76:11379-11386. - PMC - PubMed
    1. Buchholz. U. J., S. Finke, and K. K. Conzelmann. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73:251-259. - PMC - PubMed
    1. Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J. Virol. 67:4822-4830. - PMC - PubMed
    1. Delenda, C., S. Hausmann, D. Garcin, and D. Kolalofsky. 1997. Normal cellular replication of Sendai virus without the trans-frame, nonstructural V protein. Virology 228:55-62. - PubMed
    1. Didcock, L., D. F. Young, S. Goodbourn, and R. E. Randall. 1999. Sendai virus and simian virus 5 block activation of interferon-responsive genes: importance for virus pathogenesis. J. Virol. 73:3125-3133. - PMC - PubMed

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