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. 2007 Apr;81(7):3264-71.
doi: 10.1128/JVI.02590-06. Epub 2007 Jan 10.

Importance of the anti-interferon capacity of Sendai virus C protein for pathogenicity in mice

Atsushi Kato  1 , Katsuhiro Kiyotani, Toru Kubota, Tetsuya Yoshida, Masato Tashiro, Yoshiyuki Nagai
Affiliations

Importance of the anti-interferon capacity of Sendai virus C protein for pathogenicity in mice

Atsushi Kato et al. J Virol. 2007 Apr.

Abstract

The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K(151)A, E(153)A, and R(154)A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K(151)A, E(153)K, and R(157)L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1(-/-) mice but was cleared from those of STAT1(+/+) mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.

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Figures

FIG. 1.
FIG. 1.
Anti-IFN capacities of mutated SeV C proteins. HeLa cells transformed with the empty plasmid (none) or with plasmids expressing the SeV C protein (C) or the mutated Cm5, Cm*, or Cm** protein were pretreated with various amounts (0 to 103 IU/ml) of IFN-β for 24 h and then challenged with VSV or left unchallenged. Anti-VSV action was assessed by the presence of cells attached to the culture well.
FIG. 2.
FIG. 2.
Mutant SeV Cm* created from the plasmid. (A) Viral P, V, and C proteins expressed from the P gene. LLCMK2 cells inoculated with either SeV Wt or SeV Cm* were collected at 12 h postinfection. The cell lysates were immunoblotted with anti-P, anti-V, or anti-C serum. Anti-P serum reacted with the P and V proteins due to their P-common N terminus. Detected proteins are marked (left). (B) Plaques of parental SeV Wt and mutant SeV Cm* in CV1 cells.
FIG. 3.
FIG. 3.
IFN antagonism of mutant SeV Cm*. The effect of preinfection with parental SeV Wt or mutant SeV Cm* on the antiviral effect of IFN was demonstrated (top). HeLa cells treated with various amounts (0 to 103 IU/ml) of IFN-β for 12 h were subsequently challenged with VSV. Antiviral activities of IFN-β were shown by the inhibition of synthesis of VSV-specific N, P, and M proteins. Mock, cells without preinfection.
FIG. 4.
FIG. 4.
Replication of mutant SeV Cm* in 2fTGH cells and STAT1-deficient U3A cells. Parental SeV Wt and mutant SeV Cm* were inoculated into 2fTGH cells (left) and into STAT1-deficient U3A cells (right) at an MOI of 5. Cells were collected at the indicated times (hours). (Top) Cell lysates were immunoblotted with a mixture of anti-STAT1 and anti-STAT2 antibodies, or with anti-SeV or anti-C serum. Detected proteins are marked (left). (Middle) Titers of SeV Wt (open circles) or SeV Cm (closed circles) in the culture fluid. (Bottom) IFN-β produced in the culture fluid was assayed by ELISA. Open bars, SeV Wt; closed bars, SeV Cm*.
FIG. 5.
FIG. 5.
Replication and pathogenicity of mutant SeV Cm* in wild-type and STAT1 knockout mice. Five-week-old 129S6 (left) and 129S6 STAT1−/− (right) mice were infected intranasally with107 CIU of SeV Wt or SeV Cm*. Mice were weighed daily, and two or three mice of each group were sacrificed at the intervals indicated and examined for lung consolidation and virus infectivity in the lung.
FIG. 6.
FIG. 6.
Lesions in mouse lungs inoculated with mutant SeV Cm*. (A) STAT1 deficiency of 129S6 mice. Tissue lysates of mice were immunoblotted with anti-mouse STAT1 or STAT2 antibody. (B) Lungs harvested from a STAT1−/− mouse and a parental 129S6 mouse at 9 days postinoculation of SeV Cm*.
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