Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

Abstract

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

Genomic Characterization of Yogue, Kasokero, Issyk-Kul, Keterah, Gossas, and Thiafora Viruses: Nairoviruses Naturally Infecting Bats, Shrews, and Ticks

“…45 Triple-membrane-spanning NSm proteins are encoded in the M segments of orthobunyaviruses and have been implicated as scaffold proteins involved in virus assembly and morphogenesis. [46][47][48] Although also located between the Gn and Gc glycoproteins, they share no detectable sequence identity with nairovirus double-membrane-spanning NSm proteins.…”

Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

“…For orthobunyaviruses such as BUNV, viable mutants were generated by partial deletion of the central domains (27). The N-terminal region of BUNV NSm is required for virus assembly, while the C-terminal hydrophobic domain probably has a function as an internal signal sequence for the Gc glycoprotein (27). Our rSBV⌬NSm mutant also displays a large deletion within the central region of the M-segment-encoded nonstructural protein.…”

“…Because NSm-coding strategies of phleboviruses differ from those of the orthobunyaviruses, viable RVFV mutants completely lacking the NSm-coding region could be successfully rescued. For orthobunyaviruses such as BUNV, viable mutants were generated by partial deletion of the central domains (27). The N-terminal region of BUNV NSm is required for virus assembly, while the C-terminal hydrophobic domain probably has a function as an internal signal sequence for the Gc glycoprotein (27).…”

“…It is a small transmembrane protein which is colocalized with the two viral glycoproteins Gn and Gc in the Golgi complex and is probably a scaffold protein involved in virus assembly and morphogenesis. In these processes, the N-terminal part of BUNV NSm is essential, while the C terminus is dispensable (27). However, for Rift Valley fever virus (RVFV), a mosquito-transmitted phlebovirus (another genus within the family Bunyaviridae), lacking NSm was highly immunogenic, but it was only slightly attenuated and retained the ability to induce lethal hepatic and neurological disease in rats (28).…”

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

“…These NS M proteins have been shown to accumulate in the Golgi compart-ment along with the viral glycoproteins, pointing to a possible role for NS M in virus assembly. Indeed, in Bunyamwera virus, deletions of the entire NS M or its N-terminal regions prevented the plasmid rescue of virus by reverse genetics (34). A similar NS M protein has been proposed to exist for CCHFV (18,31).…”