Briefing Document: NSm Protein's Critical Role in Bunyavirus Transmission

Citation: Terhzaz, S., Kerrigan, D., Almire, F. et al. NSm is a critical determinant for bunyavirus transmission between vertebrate and mosquito hosts. Nat Commun 16, 1214 (2025). https://doi.org/10.1038/s41467-024-54809-7

Date: Received - 03 May 2024 | Accepted - 21 November 2024 | Published - 31 January 2025

Prepared for: Saleh Lab

Key Themes:

• NSm as a Unique Feature of Vector-Borne Bunyaviruses: The study establishes a strong correlation between the presence of the non-structural protein NSm and the ability of bunyaviruses to be transmitted by arthropod vectors (arboviruses). Non-arboviral bunyaviruses do not encode NSm.
• NSm's Dispensability in Monolayer Cell Cultures: While essential for in vivo transmission, NSm is shown to be largely dispensable for viral replication in both mammalian and mosquito cell lines in vitro.
• NSm's Crucial Role in Mosquito Midgut Infection and Dissemination: The research demonstrates that NSm is absolutely required for Bunyamwera virus (BUNV) to successfully infect the female Aedes aegypti mosquito following a blood meal. Specifically, it is critical for cell-to-cell spread and egress from the mosquito midgut, a major barrier to viral infection.
• Midgut-Specific Requirement: The necessity of NSm is specific to the midgut. When this barrier is bypassed through intrathoracic injection, the NSm deletion mutant (BUN-ΔNSm) can effectively infect other mosquito tissues, including the salivary glands, and be transmitted in saliva.
• NSm Facilitates Infection Foci Formation: In the midgut, BUNV-wt infection leads to the formation of large clusters of infected cells (infection foci), while BUN-ΔNSm remains confined to initially infected cells and fails to spread.
• NSm Acts Early and Transiently at the Periphery of Infection Foci: Immunocytochemistry reveals that NSm protein is localized mainly at the periphery of infection foci in the midgut, suggesting an early and transient role in spreading to neighboring cells. It is also found on the surface of infected cells and at cell junctions.
• Convergent Evolution of NSm: Phylogenetic analysis suggests that NSm has likely been acquired independently multiple times during the evolution of different bunyavirus families, indicating convergent evolution driven by the selective pressure of vector-borne transmission.
• Implications for Vaccine Development: The identification of NSm as a critical determinant for mosquito infection and transmission suggests that viruses lacking NSm could be promising candidates for live-attenuated vaccines against mosquito-borne bunyaviruses, as they would have a reduced risk of spillover in vectors.

Most Important Ideas and Facts:

• Exclusive Presence in Arboviruses: "Interestingly, this revealed a strong correlation between the presence of NSm and bunyaviruses vectored by arthropods (Fig. 1). Indeed, NSm is present in arbo-bunyaviruses only and all bunyaviruses transmitted by arthropods encode an NSm protein..." This highlights NSm as a potential signature for vector-borne transmission within the Bunyavirales order.
• Dispensable for In Vitro Replication: "Across all the cell lines tested (Fig. S2b), the growth curves of BUNV wild type (BUNV-wt) and BUN-ΔNSm were not significantly different, showing that NSm is not required for viral repli-cation in mosquito cells." This indicates that NSm's function is specific to the in vivo environment of the mosquito.
• Essential for Midgut Infection and Dissemination: "Taken together, these results revealed the crucial role of BUNV NSm in vivo as the deletion of NSm alone was sufficient to nearly abolish midgut infection and dissemination in the Ae. aegypti mosquito." This is the central finding of the study, establishing NSm's role in overcoming midgut barriers.
• Midgut Bypass Eliminates NSm Requirement: "...we did not observe differences in the salivary glands at 3 and 9dpi, indicating that both BUNV-wt and BUN-ΔNSm equally disseminated to and infected the salivary glands, when bypassing the midgut barrier through intra-thoracic inoculation." This confirms that NSm's primary function in vivo is related to traversing the midgut.
• Failure of Cell-to-Cell Spread in ΔNSm Virus: "By contrast, only a small number of individual midgut cells were positive for the viral N antigen at 72 h pbm after BUN-ΔNSm infection, showing that BUN-ΔNSm stays confined in the initially infected cells and does not spread to neighbouring cells to form large foci." This pinpoints the specific stage of infection where NSm is required.
• Rescue of ΔNSm Phenotype by NSm Expression: "Taken together, our data show that the ectopic expression of the NSm gene significantly res-cued BUN-ΔNSm infection in Ae. aegypti, demonstrating that BUNV NSm is a crucial viral determinant necessary for the successful spread of infection in the mosquito midgut." This provides strong evidence that the observed defects are directly attributable to the absence of NSm.
• NSm Localization at Infection Foci Periphery: "...the NSm protein was detected mainly at the periphery of foci (Fig. 7c–f, Fig. S5a, b), showing higher levels of expression in newly infected cells in these clusters." This suggests a dynamic role for NSm during the spread of infection.
• Convergent Evolution Implication: "As NSm is present in bunyaviruses belonging to only a few viral genera, and which are dispersed across phylogenetically distinct families12, the 'law of parsimony' supports a convergent evolution with independent and repeated acquisitions of NSm in bunyaviruses." This highlights the evolutionary importance of NSm for arboviral transmission.

Quotes:

• "Here we show that only vector-borne bunyaviruses express a non-structural protein, NSm, whose function has so far remained largely elusive."
• "Using as experimental system Bunyamwera virus (BUNV) and its invertebrate host, Aedes aegypti, we show that NSm is dis-pensable for viral replication in mosquito cells in vitro but is absolutely required for successful infection in the female mosquito following a blood meal."
• "More specifically, NSm is required for cell-to-cell spread and egress from the mosquito midgut, a known barrier to viral infection."
• "Notably, the requirement for NSm is specific to the midgut; bypassing this barrier by experimental intrathoracic infection of the mosquito eliminates the necessity of NSm for virus spread in other tissues, including the salivary glands."
• "Overall, we unveiled a key evolutionary process that allows the transmission of vector-borne bunyaviruses between arthropod and vertebrate hosts."
• "Our study reveals that NSm was acquired during the evolution of vector-borne bunyaviruses, and it is a critical determinant allowing virus transmission between vertebrate and arthropod hosts."
• "Thus, our findings demonstrate that the NSm protein is involved in cell-to-cell spread specifically in themidgut and it is therefore a key viral determinant for successful infection of bunya-viruses transmitted via a blood meal."
• "Since live-attenuated vaccine strains against arboviruses should not spillover in the vectors... the elucidation of the function of NSm as a crucial viral determinant for mosquito...