NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture

Abstract

Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.

FIG. 1.

Schematic representation of the MP-12 antigenomic-sense M segment and sequences of the pre-Gn region sections. Five in-frame translation initiation codons in the pre-Gn region are illustrated by five short vertical lines. Regions that encode the NSm and 78-kDa proteins are represented by two boxes at the top of the diagram. The sequences around the first and second AUGs in the pre-Gn region are shown at the bottom. Nucleotide substitutions in arMP-12-delNSm-1 and arMP-12-delNSm-2 at the second AUG were, respectively, GUG and GCC. An EcoRI sequence was introduced into the first AUG in arMP-12-del78. arMP-12-delNSm/78 had mutations at both the first and second AUGs as indicated.

FIG. 2.

Plaque phenotype and protein expression of mutant viruses. (A) Vero E6 cells were infected with arMP-12 and its mutant viruses as indicated. Plaques were stained with crystal violet at 3 days p.i. (B) Vero E6 cells were mock infected (Mock) or independently infected with the indicated viruses at an MOI of 1, and cell extracts were prepared using lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] in phosphate-buffered saline) at 24 h p.i. Viral proteins were separated by 12% SDS-polyacrylamide gel electrophoresis. Western blot analysis was performed using anti-NSm antibody to demonstrate NSm and 78-kDa proteins. The asterisk represents a protein of unknown origin, which was recognized by anti-NSm antibody. (C) Vero E6 cells were mock infected (Mock) or infected with the indicated viruses at an MOI of 1, and the cells were labeled with 100 μCi/ml of Tran³⁵S-label for 30 min at 8 h p.i. MP-12-specific N, Gn, and Gc proteins were immunoprecipitated using anti-N polyclonal antibody (anti-N), anti-Gn monoclonal antibody (anti-Gn), and anti-Gc monoclonal antibody (anti-Gc), respectively. Anti-Gn antibody also efficiently precipitated the 78-kDa protein (square dots). Anti-RVFV antibody (anti-RVFV) (4) was used to immunoprecipitate Gn, Gc, and N proteins of arMP-12 and its mutant viruses. Normal mouse serum (Normal serum) was used as a control. Precipitated proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis. (D) Vero E6 cells were mock infected (Mock) or independently infected with indicated viruses at an MOI of 1, and cell extracts were prepared at 8 h p.i. Western blot analysis was performed using anti-Gn monoclonal antibody, anti-Gc monoclonal antibody, and antiactin antibody to detect Gn protein, Gc protein, and actin, respectively.

FIG. 3.

Growth curves of arMP-12 and its mutant viruses. Vero (A), C6/36 (B), and MRC5 (C and D) cells were infected with arMP-12 and mutant viruses at an MOI of 1 (A, B, and C) or 0.01 (D), and the culture supernatants were collected at various times p.i. Virus titers were determined by plaque assay of Vero E6 cells.

FIG. 4.

Competition-propagation assay. (A) Shown at the top are the structures of the 5′ end of the antigenomic-sense M segment of arMP-12 and that of the arMP-12-delNSm/78 and binding sites of two primers, M18F (5′-ACACAAAGACGGTGCATT-3′) and M159R (5′-GTGAATCCCAAGCTCCTTCAAT-3′). EcoRI digestion of the arMP-12-delNSm/78-derived PCR product, but not of the arMP-12-derived PCR product, generated 140- and 19-bp-long PCR fragments (bottom). (B) A competition-propagation assay was performed as described in the text. Vero E6 cells were mock infected (Mock), independently infected with arMP-12 (arMP-12) or arMP-12-delNSm/78 (delNSm/78), or coinfected with arMP-12 and arMP-12-delNSm/78 at the indicated ratio (P₀), with virus samples passaged three times (P₃) or five times (P₅). For extraction of intracellular RNAs, viruses were infected at an MOI of 1, while virus passage was performed at an MOI of 0.1. Intracellular RNA was extracted at 8 h p.i. by using TRIzol reagent (Invitrogen). After DNase I digestion of the samples, cDNA was synthesized using random hexamers and superscript II reverse transcriptase (Invitrogen) at 42°C for 1 h. The 5′ end of the antigenomic-sense M segment was amplified from these cDNAs and plasmids pPro-T7-avM(+) [pT7-avM(+)] and pPro-T7-avM(+)-EcoRI [pT7-avM(+)-EcoRI] with primer set M18F/M159R and an Expand high-fidelity PCR system (Roche Applied Science). PCR was performed at 95°C for 3 min, followed by 30 cycles of 95°C for 40 s, 55°C for 1 min, and 72°C for 30 s. The PCR products were digested with EcoRI, and the samples were analyzed by 2% agarose gel electrophoresis.