Roles of nonstructural protein nsP2 and Alpha/Beta interferons in determining the outcome of Sindbis virus infection

Abstract

Alphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-alpha/beta) system or when IFN activity is neutralized by anti-IFN-alpha/beta antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.

FIG. 1.

Schematic representation of the double subgenomic viral genomes and ability of viral mutants to form plaques on BHK-21 and NIH 3T3 cells. In all of the genomes, the first subgenomic promoter was driving the expression of GFP and the second one was driving the expression of SIN structural genes derived from the TE12 strain of SIN. The SIN/G variant was different from wtSIN by one amino acid in nsP2, P₇₂₆→G. The SIN44 differed from wtSIN by clustered mutations in nsP1, which did not change the encoded protein sequence but destroyed the secondary structure of the replicational enhancer, the 51-nt CSE. Viral stocks generated by electroporation of in vitro-synthesized RNAs were titrated simultaneously on BHK-21 and NIH 3T3 cells. Plaques were allowed to develop for 48 h prior to fixation and staining. Stained, infected monolayers of both cell types correspond to the same dilutions of viruses.

FIG. 2.

Analysis of the replication and transcription of SIN RNAs in infected cells. NIH 3T3 cells in six-well Costar plates were infected with different SIN viruses at an MOI of 20. At 1 h (lanes 1 to 3) and 5 h (lanes 4 to 6) p.i., medium in the wells was replaced by 1 ml of alpha MEM supplemented with 10% FBS, dactinomycin (2 μg/ml), and [³H]uridine (20 μCi/ml). After 3 h of incubation at 37°C, RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes: 1 and 4, RNAs isolated from the cells infected with wtSIN; 2 and 5, RNAs from the cells infected with SIN44; 3 and 6, RNAs from the cells infected with SIN/G.

FIG. 3.

Analysis of virus growth and protein synthesis in virus-infected cells. NIH 3T3 cells were infected with wtSIN, SIN/G, and SIN44 at an MOI of 20 PFU/cell. (A) At the indicated times, media were replaced and virus titers were determined as described in Materials and Methods. In other wells, proteins were pulse-labeled with [³⁵S]methionine and analyzed on sodium dodecyl sulfate-10% polyacrylamide gel. Gels were dried and autoradiographed (B) or analyzed on a Storm 860 PhosphorImager (C). The levels of synthesis of cellular proteins were determined by measuring radioactivity in the protein band corresponding to actin (indicated by the arrow) and normalized to radioactivity in the actin band in uninfected cells.

FIG. 4.

The results of GeneChip analysis of mRNA expression in uninfected NIH 3T3 cells and the cells infected with wtSIN and SIN/G viruses at an MOI of 20 PFU/cell. The RNAs were isolated at 17 h p.i., and the analysis was performed as described in Materials and Methods. LPS, lipopolysaccharide.

FIG. 5.

Virus growth and IFN-α/β production by the NIH 3T3 cells infected with wtSIN, SIN/G, and SIN44 variants at MOIs of 0.004 (A) and 20 (B) PFU/cell. Virus titers and concentrations of IFN-α/β were determined in the same samples harvested at the indicated time points. These data represent one of a series of repeated experiments which generated very reproducible data.

FIG. 6.

Virus growth and IFN-α/β production by the NIH 3T3 cells infected with the SIN/G mutant at an MOI of 20 PFU/cell. Medium was replaced at the indicated time points; virus titers and concentrations of IFN-α/β were determined as described in Materials and Methods and demonstrate the accumulation of IFN and virus in the periods between the indicated time points. These data represent one of three repeated experiments which generated reproducible data.

FIG. 7.

IFN-α/β sensitivity assay. Monolayers of NIH 3T3 cells in 12-well plates were treated with the indicated concentrations of murine IFN-α/β for 24 h and then infected with wtSIN, SIN/G, and SIN44 variants at an MOI of 20. Titers of released viruses were measured 24 h p.i.. The results were normalized to titers of viruses released from the cells not treated with IFN-α/β.

FIG. 8.

(A) Growth of wtSIN and SIN/G viruses in MEFs derived from IFN-α/βR^−/− mice; (B) growth of SIN/G virus in NIH 3T3 cells in the presence and absence of anti-IFN-α/β antibodies (+AB and −AB, respectively). Cells were infected at an MOI of 20 PFU/cell, and media were replaced at the indicated time points to determine viral titers. Sheep anti-mouse IFN-α/β AB was present in the medium at a concentration of 1,000 IFN neutralization units per ml. These data represent one of two repeated experiments.

FIG. 9.

(A) Survival of mice infected with wtSIN, SIN/G, and SIN44; virus growth (B) and production of IFN-α/β (C) in mouse brains. Two-day-old CD1 mice were inoculated intracranially with 10³ PFU of the indicated virus and observed for 15 days. Virus titers and concentrations of IFN-α/β were determined as described in Materials and Methods. Error bars, standard deviations.