Analysis of NS2-dependent effects on influenza PB1 segment extends replication requirements beyond the canonical promoter

TL;DR

This study shows that influenza NS2 affects replication of viral segments beyond the standard promoter regions.

Contribution

The study identifies NS2-dependent replication requirements in PB1 beyond the canonical promoter sequences.

Findings

NS2 alters replication sequence requirements beyond U12 and U13 in PB1.

Mutant libraries reveal NS2's role in segment-specific replication dynamics.

Conserved regions outside U12 and U13 are involved in NS2-dependent replication.

Abstract

Influenza A virus encodes conserved promoter sequences. Using minimal replication assays—transfections with viral polymerase, nucleoprotein, and a genomic template—these sequences were identified as 13nt at the 5’ end of the genomic RNA (U13) and 12nt at the 3’ end (U12). Other than the fourth 3’ nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules of the segmented influenza A genome. However, individual segments can exhibit different dynamics during infection. Influenza NS2, which modulates transcription and replication differentially between genomic segments, may provide an explanation. Here, we assess how internal sequences of two genomic segments, HA and PB1, contribute to NS2-dependent replication and map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequences, providing a potential mechanism for segment-specific replication dynamics across the influenza genome.

The eight genomics segments of Influenza A virus exhibit different replication dynamics during infection. Here, the authors show through a range of mutant libraries of HA and PB1 segments that Influenza NS2 regulates the replication of viral segments via conserved regions outside the U12 and U13 promoters.

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