The N Terminus of the Influenza B Virus Nucleoprotein Is Essential for Virus Viability, Nuclear Localization, and Optimal Transcription and Replication of the Viral Genome

Abstract

The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication. IMPORTANCEThe multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus contains an evolutionarily conserved 50-amino-acid N-terminal extension that is absent from the NP of influenza A viruses. In this study, we show that this N-terminal extension is essential for virus viability, and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore, we demonstrate for the first time that the N terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension, these viruses may have evolved separate mechanisms to regulate these processes.

Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

“…This length of time is sufficient for the influenza virus to develop a type-specific function as exemplified by type-specific virus-host interactions in NS1 [ 11 , 12 ]. Furthermore, conservation of protein function does not necessarily support that sequence conservation exists at the primary sequence level, which is evidenced by the differences between the nuclear localization signal of influenza A and B NP proteins [ 82 , 83 ]. In fact, this study reveals that type-specific functional residues are prevalent in the influenza virus PA protein.…”

Knowns and Unknowns of Influenza B Viruses

“…Different mutations in the N-terminus affected the ability to rescue virus or viral replication and transcription. Moreover, the entire Nterminus appears to be essential for optimal localization in the nucleus during infection and residues 51-81 were suggested to contain a bipartite NLS [79].…”

The Functional Study of the N-Terminal Region of Influenza B Virus Nucleoprotein

“…Hela cells(ATCC CCL2)[ 15 ], Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) [ 14 , 16 , 17 ] and HEK 293T/17 cells (ATCC CRL-11268)[ 10 , 11 ] were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. All cells were incubated at 37°C in 5% CO2.…”

“…It is known that the N-terminal of BNP plays a role in the transcription and replication of viral RNP, possibly by nuclear targeting (aa 44–47) and proteomic protection (aa 1–15)[ 13 , 14 ]. However, whether it is involved in other major functions of BNP, such as oligomerization or RNA binding, have not been explored.…”