The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection

Abstract

Rift Valley fever (RVF) virus belongs to the Bunyaviridae family of segmented negative-strand RNA viruses and causes mosquito-borne disease in sub-Saharan Africa. We report the development of a T7 RNA polymerase-driven plasmid-based genetic system for the virulent Egyptian isolate, ZH501. We have used this system to rescue a virus that has a 387 nucleotide deletion on the genomic M segment that eliminates the coding region for two non-structural proteins known as NSm. This virus, DeltaNSm rZH501, is indistinguishable from the parental ZH501 strain with respect to expression of structural proteins and growth in cultured mammalian cells.

Figure 1

Sequencing of 3′ terminus and NSm region of the genomic-sense M segment of ZH501 and ΔNSm rZH501 viruses. A) Nucleotide and amino acid sequence for the NSm region of ZH501strain. The translation is in single letter code above the nucleotide sequence and numbering refers to the nucleotide position. The methionine (M) residues are in bold type. NSm1 and NSm2 begin at the first and second methionine residues, respectively. The nucleotides absent in the ΔNSm rZH501 virus are underlined. Translation of the polyprotein precursor that yields only Gn and Gc initiates at the fourth methionine residue. The UTR (nucleotides 1–21) of the ΔNSm rZH501 virus is identical to that of the ZH501 strain. B) RT-PCR products that include nucleotides 1–890 of the M segment (numbering relative to the ZH501 strain in the anti-genomic sense) were obtained by 3′ RACE. Analysis of the resulting sequence demonstrates that the M segment of ΔNSm rZH501 has the expected deletion of nucleotides 22–408.

Figure 2

Viral protein expression in ZH501 and ΔNSm rZH501-infected cells. A) Extracts from ZH501, ΔNSm rZH501 and mock-infected cells were immunoblotted with antibodies that recognize all structural proteins (anti-RVF virus HMAF). The position and size of the molecular weight markers and position and identity of the viral proteins are indicated.

Figure 3

ΔNSm rZH501-infected cells are indistinguishable from that of ZH501-infected cells. Vero E6 cells were infected with either ΔNSm rZH501 or ZH501 viruses, then fixed with formalin at 24 h-post-infection. Cells were subsequently stained with monoclonal antibodies that recognize either N or Gn, followed by fluorescently labeled anti-mouse secondary antibodies.

Figure 4

ZH501 and ΔNSm rZH501 viruses have similar growth rates. Monolayers of Vero E6 cells were infected at an MOI of 1 or 0.1, supernatants were harvested at the indicated times and analyzed by plaque assay. The concentration of virus in the supernatant is expressed in plaque forming units (PFU) per milliliter.