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 competitionpropagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.Rift Valley fever virus (RVFV) (of the genus Phlebovirus, family Bunyaviridae), which is the cause of severe epidemics in ruminants of sub-Saharan African, is also recognized as a human pathogen that can cause a syndrome with fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (1, 9, 10). RVFV has a single-stranded, tripartite RNA genome composed of the L, M, and S segments. The L segment encodes the L protein, an RNA-dependent RNA polymerase. Using an ambisense strategy, the S segment expresses the N protein and the nonstructural NS proteins (12).The RVFV M segment encodes four proteins, two major envelope glycoproteins, Gn (or G2) and Gc (or G1), which most probably bind to an as-yet-unknown viral receptor molecule to initiate virus infection, and two minor proteins, the 14-kDa nonstructural NSm protein (7) and the 78-kDa protein, which is reported to be a structural protein (15). The biological functions of the NSm and 78-kDa proteins are totally unknown, but they probably do not have a role in viral RNA synthesis; RVFV minigenome RNA replication and transcription occur efficiently in the absence of expression of the NSm, 78-kDa, and Gn and Gc proteins (2). The region upstream from the Gn gene (pre-Gn region) contains five in-frame AUG codons (Fig. 1), and it appears that each of these five AUGs serves as an initiation codon of different proteins. The first and second AUGs serve as initiation codons for the 78-kDa and NSm proteins, respectively, while the third, fourth, and fifth AUGs initiate the Gn-Gc fusion protein (6, 13, 16). The 78-kDa protein consists of pre-Gn and Gn regions. NSm contains the region that starts from the second AUG to the end of the pre-Gn region. A precursor of the Gn-Gc fusion protein is translated from the third to the fifth AUG, and then it undergoes protein processing to generate Gn and Gc proteins. Gn and Gc protein synthesis still occurs in the absence of the first and second AUGs (7, 16).To know whether the NSm and 78-kDa proteins are required for RVFV replication, we attempted to generate mutant viruses lacking the expression of one or both proteins by using a reverse-genetics system of an attenuated vaccine candidate of RVFV, namely MP-12 (3). Previously, we reported that MP-12 recovered by using a reverse-genetics system carries an XhoI site in each RNA segment (3), yet the rescued parental ...

Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells

“…ϭ 3). In the case of infections with the NSs and NSm mutant viruses (6,28), cells were infected with the appropriate mutant constructs (m.o.i. ϭ 3).…”

“…Additionally, the M segment encodes a 78-kDa protein of unknown function and a small nonstructural protein, NSm. NSm has been demonstrated to have an antiapoptotic function in infected cells (5,6). The S (small) segment encodes the N protein and a second nonstructural protein, NSs.…”

Nuclear Relocalization of Polyadenylate Binding Protein during Rift Valley Fever Virus Infection Involves Expression of the NSs Gene

“…The glycoproteins are essential for attachment and entry, as well as assembly (14). While NSm is dispensable for replication in cell culture and animal models, it has been shown to inhibit apoptosis in infected cells (15)(16)(17)(18). Additionally, NSm has been shown to be important for RVFV infection and transmission in mosquitoes (19).…”

Identification of a Novel C-Terminal Cleavage of Crimean-Congo Hemorrhagic Fever Virus PreG N That Leads to Generation of an NS M Protein

“…For the phleboviruses and orthobunyaviruses, however, relatively little is known about the function of their NS M proteins. The RVFV NS M 1 protein can be coimmunoprecipitated with the G C glycoprotein, though NS M -deficient RVFV replicates in cultured mammalian cells with no apparent growth defects (19,43). For Orthobunyavirus genus members, the NS M proteins are known to traffic to the Golgi compartment, where they may interact with the glycoproteins and nucleoprotein (34).…”

“…Ideally, the function of the NS M protein would best be addressed in the context of infections with recombinant CCHFV lacking the NS M or possessing mutations within it, as has been recently done for BUNV (34), Maguari virus (30), and RVFV (19,43). Although a reverse genetics system exists for CCHFV (15), it requires superinfection with wild-type CCHFV to drive expression of plasmid-based minigenomes.…”