Mapping of the influenza A virus genome RNA structure and interactions reveals essential elements of viral replication.

Influenza A virus (IAV) represents a constant public health threat. The single-stranded, segmented RNA genome of IAV is replicated in host cell nuclei as a series of 8 ribonucleoprotein complexes (vRNPs) with RNA structures known to exert essential function to support viral replication. Here, we investigate RNA secondary structures and RNA interactions networks of the IAV genome and construct an in vivo structure model for each of the 8 IAV genome segments. Our analyses reveal an overall in vivo and in virio resemblance of the IAV genome conformation but also wide disparities among long-range and intersegment interactions. Moreover, we identify a long-range RNA interaction that exerts an essential role in genome packaging. Disrupting this structure displays reduced infectivity, attenuating virus pathogenicity in mice. Our findings characterize the in vivo RNA structural landscape of the IAV genome and reveal viral RNA structures that can be targeted to develop antiviral interventions.

The Hierarchical Sequence Requirements of the H1 Subtype-Specific Noncoding Regions of Influenza A Virus.

The genome of influenza A virus consists of eight single-stranded viral RNA (vRNA) segments. The nonconserved noncoding regions (NCRs) at the 3' and 5' termini of each segment show extremely low divergence and mutation rate. They appear as segment specific among the eight segments and also subtype specific among different subtype-determinant hemagglutinin (HA) and neuraminidase (NA) segments. In order to acquire in-depth knowledge on the sequence requirements of the segment-specific or subtype-specific NCRs (ssNCRs), we, in the context of WSN (H1N1) reverse genetics, designed a virus random nucleotide selection assay (vRNSA) in which we generated pHW2000-HA plasmid libraries with random nucleotides in each grouped nucleotide positions in the 3' and 5' H1-ssNCRs, followed by virus rescue, serial passage, and deep sequencing. The resulting sequence logos present a visualized dynamic overview of the hierarchical sequence requirements of the 3' and 5' H1-ssNCRs. It showed that, in the process of continuous passage, the 3' H1-ssNCR, in general, stabilized more quickly than the 5' H1-ssNCR. The nucleotides close to the highly conserved 3' and 5' promoter regions showed higher sequence stringency than nucleotides away from the promoter regions. All stabilized sequences displayed a common feature of high A/U ratios. Especially with our mutational function analyses, we demonstrate that the 3' promoter-proximal nucleotides could cooperatively exert a direct effect on the transcription and replication of the HA segment. Together, these results provide in-depth knowledge for understanding the NCRs of influenza A virus. IMPORTANCE The segment-specific and subtype-specific nonconserved noncoding regions (ssNCRs) at both 3' and 5' ends of viral RNA segments of influenza A virus are largely conserved among the same segments of different viruses. However, the function-related sequence requirements of these ssNCRs remain unclear. In this study, through a novel self-designed vRNSA approach, we present a visualized dynamic overview diagram directly reflecting the hierarchical sequence requirements within and between the 3' and 5' H1-ssNCRs. The in-depth functional mutagenesis analyses further revealed that specific nucleotides in the 3' promoter-proximal region could cooperatively exert a direct effect on viral RNA transcription and replication. This work further advanced our knowledge in understanding the nonconserved noncoding regions of influenza A viruses.

Synergistic Effect between 3'-Terminal Noncoding and Adjacent Coding Regions of the Influenza A Virus Hemagglutinin Segment on Template Preference.

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding hemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype specific, varying significantly in sequence and length at both the 3' and 5' termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3'-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable, and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multisegment environment. These results provide novel insights into the role of the 3'-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3'-end H1-ssNCR and observed a synergistic effect between the 3'-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of the HA segment on template preference in a multisegment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

A reverse genetics system for enterovirus D68 using human RNA polymerase I.

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.