Question

In what three ways can influenza viruses solve the problem of expressing multiple proteins with limited genetic space

Answer 1

Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity.

Rearrangement of the influenza virus genome was accomplished by expressing the NEP/NS2 protein from a single polypetide downstream of the PB1 gene. Foreign genes of interest were cloned downstream of a full-length or truncated NS1 gene between two AarI cloning sites, so that there was no introduction of exogenous sequences. Three nucleotide mutations were introduced into the full-length NS1 by site-directed mutagenesis to prevent residual splicing and/or NS2 expression. The splicing donor site was modified from G to A at position 56, and a stop codon was inserted early in NS2 and out of frame with NS1 via a C548A mutation.

Influenza A viral replication peaks approximately 48 hours after inoculation into the nasopharynx and declines slowly, with little virus shed after about six days. The virus replicates in both the upper and lower respiratory tract. Even after the infectious virus can no longer be recovered, viral antigen can be detected in cells and secretions of infected individuals for several days.

The diagnosis of influenza can be established by viral culture, demonstration of viral antigens, or demonstration of viral genetic material (in clinical specimens), or rises/falls in specific antibody titers in serum or respiratory secretions

One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus hemagglutinin protein—the primary antibody target of the immune system—on its evolution. Using a combination of computational estimates of stability and experiments, our analysis found that viruses with more-stable hemagglutinin proteins were associated with long-term persistence in the population. There are two potential reasons for the observed persistence. One is that more-stable proteins tolerate destabilizing mutations that less-stable proteins could not, thus increasing opportunities for immune escape. The second is that greater stability increases the fitness of the virus through increased production of infectious particles. Further research on the relative importance of these mechanisms could help inform the annual influenza vaccine composition decision process