Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein

Abstract

Viruses in the genus Orthobunyavirus, family Bunyaviridae, have a genome comprising three segments (called L, M, and S) of negative-sense RNA. Serological studies have classified the >170 named virus isolates into 18 serogroups, with a few additional as yet ungrouped viruses. Until now, molecular studies and full-length S-segment nucleotide sequences were available for representatives of eight serogroups; in all cases, the S segment encodes two proteins, N (nucleocapsid) and NSs (nonstructural), in overlapping open reading frames (ORFs) that are translated from the same mRNA. The NSs proteins of Bunyamwera virus (BUNV) and California serogroup viruses have been shown to play a role in inhibiting host cell mRNA and protein synthesis, thereby preventing induction of interferon (IFN). We have determined full-length sequences of the S segments of representative viruses in the Anopheles A, Anopheles B, and Tete serogroups, and we report here that these viruses do not show evidence of having an NSs ORF. In addition, these viruses have rather longer N proteins than those in the other serogroups. Most of the naturally occurring viruses that lack the NSs protein behaved like a recombinant BUNV with the NSs gene deleted in that they failed to prevent induction of IFN-beta mRNA. However, Tacaiuma virus (TCMV) in the Anopheles A serogroup inhibited IFN induction in a manner similar to that of wild-type BUNV, suggesting that TCMV has evolved an alternative mechanism, not involving a typical NSs protein, to antagonize the host innate immune response.

FIG. 1.

ORF analysis of Orthobunyavirus S segments. Sequences were analyzed using DNAMAN software (Lynnon corporation). Each reading frame is shown on a separate line, with ticks above the line indicating AUG codons and ticks below the line indicating translational stop codons. The boxed areas represent the N and NSs overlapping ORFs as indicated.

FIG. 2.

Cell-free translation of proteins expressed from S-segment cDNA clones. S-segment cDNAs were cloned under the control of the T7 promoter and used to program either wheat germ or rabbit reticulocyte-coupled transcription/translation systems as directed by the manufacturer (TnT; Promega). Empty plasmid vector was included as a control. Radiolabeled proteins were separated on NuPAGE 6 to 12% gradient gels. Positions of viral N and NSs proteins are indicated.

FIG. 3.

IFN production in infected cells. (A) Induction of IFN-β mRNA. Total RNA was extracted from mock- or virus-infected cells as indicated and reverse transcribed with random hexamer primers, and then cDNA was PCR amplified using primers specific for human IFN-β mRNA (upper panel) or γ-actin mRNA (lower panel). Amplified DNA products were electrophoresed in an agarose gel and stained with ethidium bromide. (B) Induction of IFN-β promoter. A549 cells were transfected with the IFN-β promoter containing reporter plasmid and 5 h later infected with the different viruses or mock infected. Cells were lysed 16 h later, and luciferase activity was determined. The activity in mock-infected cells was taken as 1, and the activity in virus-infected cells was expressed relative to this. (C) Biological activity for IFN production. Twofold dilutions of medium from A549 cells infected with different viruses, either singly or in combination as indicated, or mock infected were used to treat fresh A549 cells in 96-well plates for 24 h. The cells were then infected with EMCV, and the development of CPE was monitored 48 h later by staining with Giemsa. m, molecular ladder.