Mutations in the M-Gene Segment Can Substantially Increase Replication Efficiency of NS1 Deletion Influenza A Virus in MDCK Cells

Abstract

bInfluenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

An NS-segment exonic splicing enhancer regulates influenza A virus replication in mammalian cells

“…It has been suggested that the viral polymerase complex is involved in regulation of M2 mRNA splicing regulation through interaction with the splice site in exon 2 and other studies have also argued that viral polymerases have a role in this process444748. Evidence from our own and other studies have suggested a mechanism involving NS1 in the regulation of splicing of M mRNAs, as the M2/M1 mRNA ratio is altered in viruses lacking the NS1 protein94449. However, it is less clear how expression of the spliced form of NS mRNA is regulated to influence NEP expression.…”

Influenza A(H1N1)pdm09 M and HA segments sequences from Rio Grande do Sul, Brazil

“…Experiment using guinea pigs demonstrated that strains without pH1N1 M could not transmit the virus while those containing NL + M + NA + HA had great transmission even with a 100 PFU virus dose (Campbell et al 2014). According to van Wielink and colleagues (2012), mutations in M and NP indicated an average decrease in virus titers when combined while mutations in M itself increased virus titer indicating that M alone determined enhanced replication. NS1 is expressed at high levels after infection and facilitates virus replication (Hale et al 2008).…”

“…2014). According to van Wielink et al . (2012), combined mutations in M and NP lead to an average decrease in virus titer, whereas mutations in M itself increase the virus titer, indicating that M alone determines enhanced replication.…”

“…One of the goals of NS1 is mRNA splicing of the M segment (Robb and Fodor 2012). The increased titer of delNS1 was due to the M1 amino acid substitution at M86V, a region near to helix six domain, which is a positively charged surface region (van Wielink et al . 2012, Sha and Luo 1997).…”

“…2012, Sha and Luo 1997). Adaptation of M-segment mutations could compensate for the absence of NS1 protein (delNS1), and these modifications can increase the expression of M1/M2 ratio, even if M2 has silent mutations and M1 with silent or nonsilent mutations that affect M1 mRNA splicing efficiency (van Wielink et al . 2012).…”