Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

Abstract

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

Temperature dependent viral tropism: understanding viral seasonality and pathogenicity as applied to the avoidance and treatment of endemic viral respiratory illnesses

“…control the expression of the proteins NS1 and NEP during influenza A infection. 68 RNA secondary structure is inherently ts, and conserved RNA structures (such as, in SARS-CoV-2, the s2m structure, the 3 0 UTR pseudoknot, and the coronavirus packaging signal) may comprise 'RNA thermometers' contributing to the thermal sensitivity of such viruses. 69 Two studies found that temperature affects the balance between transcription and viral replication.…”

“…Virologists tend to focus on mutations that change the sequences of viral proteins. However, RNA secondary structure is known to affect protein expression and has been shown to control the expression of the proteins NS1 and NEP during influenza A infection 68 . RNA secondary structure is inherently ts, and conserved RNA structures (such as, in SARS‐CoV‐2, the s2m structure, the 3′ UTR pseudoknot, and the coronavirus packaging signal) may comprise ‘RNA thermometers’ contributing to the thermal sensitivity of such viruses 69 .…”