Genome Of Influenza Research Articles

Structural insights into Influenza A virus RNA polymerase PB1 binding to nuclear import host factor RanBP5

The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly synthesized RNA polymerase subunits must be imported into the nucleus. Although several models have been proposed for this process, the consensus is that the RNA polymerase subunits PB1 and PA form a dimer in the cytoplasm and are transported into the nucleus by Ran binding protein 5 (RanBP5). The PB2 subunit undergoes separate transport to complete the nuclear import. However, the molecular mechanism of nuclear import by host factors and their interactions with proteins are largely unknown. Here we present the structural analysis of the RanBP5 and PB1 NLS domain complex by cryo-EM at 3.2 Å resolution. The pattern shows that the NLS domain of PB1 does not exist in a secondary structure and interacts with RanBP5 in a wrapped state. In addition, biochemical analyses of the mutant have identified critical amino acid sites involved in complex binding. The results suggest a stepwise assembly of influenza virus structural components regulated by nuclear import mechanisms and host factor binding, with important implications for drug discovery research.

Influenza outbreak during the surge of SARS-CoV-2 omicron in a metropolitan area from southern Brazil: genomic surveillance.

Influenza circulation was significantly affected in 2020-21 by the COVID-19 pandemic. During this time, few influenza cases were recorded. However, in the summer of 2021-22, an increase in atypical influenza cases was observed, leading to the resurgence of influenza in the southernmost state of Brazil, Rio Grande do Sul (RS). The present study aimed to identify the circulation of FLUAV, FLUBV and SARS-CoV-2 and characterize the influenza genomes in respiratory samples using high-throughput sequencing technology (HTS). Respiratory samples (n = 694) from patients in RS were selected between July 2021 and August 2022. The samples were typed using reverse transcriptase real-time PCR (RT-qPCR) and showed 32% (223/694) of the samples to be positive for SARS-CoV-2, 7% for FLUAV (H3) (49/694). FLUBV was not detected. RT-qPCR data also resulted in FLUAV and SARS-CoV-2 co-infections in 1.7% (4/223) of samples tested. Whole genome sequencing of FLUAV produced 15 complete genomes of the H3N2 subtype, phylogenetically classified in the 3C.2a1b.2a.2a.3 subclade and revealing the dominance of viruses in the southern region of Brazil. Mutation analysis identified 72 amino acid substitutions in all genes, highlighting ongoing genetic evolution with potential implications for vaccine effectiveness, viral fitness, and pathogenicity. This study underscores limitations in current surveillance systems, advocating for comprehensive data inclusion to enhance understanding of influenza epidemiology in southern Brazil. These findings contribute valuable insights to inform more effective public health responses and underscore the critical need for continuous genomic surveillance.

Hyper-Interferon Sensitive influenza induces adaptive immune responses and overcomes resistance to anti-PD-1 in murine non-small cell lung cancer.

Despite recent advances in immunotherapy with immune checkpoint inhibitors (ICI), many patients with non-small cell lung cancer (NSCLC) fail to respond or develop resistance after an initial response. In situ vaccination (ISV) with engineered viruses has emerged as a promising antigen-agnostic strategy that can both condition the tumor microenvironment (TME) and augment anti-tumor T cell responses to overcome immune resistance. We engineered a live attenuated viral vaccine, Hyper-Interferon Sensitive virus (HIS), by conducting a genome-wide functional screening and introducing eight interferon (IFN)-sensitive mutations in the influenza genome. Compared to wild-type (WT) influenza, HIS replication was attenuated in immunocompetent hosts, enhancing its potential as a safe option for cancer therapy. HIS ISV elicited robust yet transient type I IFN responses in murine NSCLCs, leading to an enrichment of polyfunctional effector Th1 CD4 and cytotoxic CD8 T cells into the tumor. HIS ISV demonstrated enhanced anti-tumor efficacy compared to WT in multiple syngeneic murine models of NSCLC with distinct driver mutations and varying mutational burden. This efficacy was dependent on host type 1 IFN responses and T lymphocytes. HIS ISV overcame resistance to anti-PD-1 in LKB-1 deficient murine NSCLC, resulting in improved overall survival and enduring systemic tumor-specific immunity. These studies provide compelling evidence to support further clinical evaluation of HIS as a novel 'off-the-shelf' ISV strategy for patients with NSCLC refractory to ICI.

Predicting host species susceptibility to influenza viruses and coronaviruses using genome data and machine learning: a scoping review.

Predicting which species are susceptible to viruses (i.e., host range) is important for understanding and developing effective strategies to control viral outbreaks in both humans and animals. The use of machine learning and bioinformatic approaches to predict viral hosts has been expanded with advancements in in-silico techniques. We conducted a scoping review to identify the breadth of machine learning methods applied to influenza and coronavirus genome data for the identification of susceptible host species. The protocol for this scoping review is available at https://hdl.handle.net/10214/26112. Five online databases were searched, and 1,217 citations, published between January 2000 and May 2022, were obtained, and screened in duplicate for English language and in-silico research, covering the use of machine learning to identify susceptible species to viruses. Fifty-three relevant publications were identified for data charting. The breadth of research was extensive including 32 different machine learning algorithms used in combination with 29 different feature selection methods and 43 different genome data input formats. There were 20 different methods used by authors to assess accuracy. Authors mostly used influenza viruses (n = 31/53 publications, 58.5%), however, more recent publications focused on coronaviruses and other viruses in combination with influenza viruses (n = 22/53, 41.5%). The susceptible animal groups authors most used were humans (n = 57/77 analyses, 74.0%), avian (n = 35/77 45.4%), and swine (n = 28/77, 36.4%). In total, 53 different hosts were used and, in most publications, data from multiple hosts was used. The main gaps in research were a lack of standardized reporting of methodology and the use of broad host categories for classification. Overall, approaches to viral host identification using machine learning were diverse and extensive.

First Case of Highly Pathogenic Avian Influenza H5N1 in Montenegro

Abstract Avian influenza, commonly known as bird flu, is a highly infectious viral disease that affects birds, including wild water birds and poultry. The emergence and spread of highly pathogenic avian influenza (HPAI) strains, such as H5N1, have raised concerns due to their potential to cause severe outbreaks and cross the species barrier, leading to human infections and global public health emergencies. In this study, we report the first case of HPAI H5N1 detection in Montenegro. Twenty-six carcasses of dalmatian pelicans were found in Skadar Lake, Montenegro, and the H5N1 subtype was confirmed through molecular testing in the samples from pelicans. The whole influenza genome was sequenced and belonging to clade 2.3.4.4b was determined.

Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection.

This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

Genome characterization of influenza A and B viruses in New South Wales, Australia, in 2019: A retrospective study using high-throughput whole genome sequencing.

During the 2019 severe influenza season, New South Wales (NSW) experienced the highest number of cases in Australia. This study retrospectively investigated the genetic characteristics of influenza viruses circulating in NSW in 2019 and identified genetic markers related to antiviral resistance and potential virulence. The complete genomes of influenza A and B viruses were amplified using reverse transcription-polymerase chain reaction (PCR) and sequenced with an Illumina MiSeq platform. When comparing the sequencing data with the vaccine strains and reference sequences, the phylogenetic analysis revealed that most NSW A/H3N2 viruses (n= 68; 94%) belonged to 3C.2a1b and a minority (n= 4; 6%) belonged to 3C.3a. These viruses all diverged from the vaccine strain A/Switzerland/8060/2017. All A/H1N1pdm09 viruses (n= 20) showed genetic dissimilarity from vaccine strain A/Michigan/45/2015, with subclades 6B.1A.5 and 6B.1A.2 identified. All B/Victoria-lineage viruses (n= 21) aligned with clade V1A.3, presenting triple amino acid deletions at positions 162-164 in the hemagglutinin protein, significantly diverging from the vaccine strain B/Colorado/06/2017. Multiple amino acid substitutions were also found in the internal proteins of influenza viruses, some of which have been previously reported in hospitalized influenza patients in Thailand. Notably, the oseltamivir-resistant marker H275Y was present in one immunocompromised patient infected with A/H1N1pdm09 and the resistance-related mutation I222V was detected in another A/H3N2-infected patient. Considering antigenic drift and the constant evolution of circulating A and B strains, we believe continuous monitoring of influenza viruses in NSW via the high-throughput sequencing approach provides timely and pivotal information for both public health surveillance and clinical treatment.

Nucleic acid-based electrochemical biosensor for detection of influenza B by gold nanoparticles.

The influenza virus is a pervasive pathogen that exhibits increased prevalence during colder seasons, resulting in a significant annual occurrence of infections. Notably, pharmaceutical interventions effective against influenza A strains often exhibit limited efficacy against influenza B variants. Against this backdrop, the need for innovative approaches to accurately and swiftly differentiate and detect influenza B becomes evident. Biosensors play a pivotal role in this detection process, offering rapid, specific, and sensitive identification of the virus, facilitating timely intervention and containment efforts. Oligonucleotide sequences targeting the conserved B/Victoria/2/87 influenza virus NP region were designed. Nasopharyngeal swabs were collected from patients suspected of influenza virus infection, and viral RNA was extracted. RNA quality was assessed through one-step PCR. cDNA synthesis was performed using random hexamers, and real-time PCR quantified the influenza genome. Gold nanoparticles were immobilized on a surface to immobilize the specific DNA probe, and electrochemical hybridization was electrochemically followed. The biosensor exhibited high selectivity and effective distinction of complementary sequences from mismatches and influenza virus cDNA genome. The biosensor successfully detected the influenza B virus genome in real samples. Non-influenza samples yielded no significant hybridization signals. The comparison between the results obtained from the biosensor and real-time PCR revealed full agreement of these methods. The biosensor utilized electrochemical detection of hybridization and proved effective in detecting the influenza B virus genome with high specificity, sensitivity, and selectivity. Comparative analysis with real-time PCR underscored the accuracy and potential applicability of the biosensor in rapid and specific virus detection. This innovative approach holds promise for future diagnostic and epidemiological applications in detecting influenza B virus and other pathogens.

Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo–electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.

ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions.

It is now established that many viruses that threaten public health establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol and reduces genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.

Contribution of the Nuclear Localization Sequences of Influenza A Nucleoprotein to the Nuclear Import of the Influenza Genome in Infected Cells.

Replication of the RNA genome of influenza A virus occurs in the nucleus of infected cells. The influenza nucleoprotein (NP) associated with the viral RNA into ribonucleoprotein complexes (vRNPs) is involved in the nuclear import of the viral genome. NP has two nuclear localization sequences (NLSs), NLS1 and NLS2. Most studies have concentrated on the role of NP's NLSs using in vitro-assembled or purified vRNPs, which may differ from incoming vRNPs released in the cytoplasm during an infection. Here, we study the contribution of the NP's NLSs to the nuclear import of vRNPs in a cell culture model system for influenza infection: human lung carcinoma cells infected with viruses containing NP-carrying mutations in NLS1 or NLS2 (NLS2MT), generated by reverse genetics. We found that cells infected with these mutant viruses were defective in the nuclear import of incoming vRNPs and produced reduced amounts of newly synthesized NP, newly assembled vRNP, and progeny virus. In addition, NLS2MT-infected cells were also defective in the nucleolar accumulation of NP, confirming the nucleolar localization role of NLS2. Our findings indicate that both NLS1 and NLS2 have to be present for successful infection and demonstrate the crucial role of these two NLSs in the infection cycle of the influenza A virus.

Local changes in viral RNA sequence drive global changes in influenza nucleoprotein binding.

The genome of influenza A viruses (IAV) consists of eight negative-sense RNA segments that are coated by viral nucleoprotein (NP). Until recently, it was assumed that NP binds viral genomic RNA (vRNA) uniformly along the entire segment. However, genome-wide studies have revised the original model in that NP instead binds preferentially to certain regions of vRNA, while others are depleted for NP binding. Even strains with high sequence similarity exhibit distinct NP-binding profiles. Thus, it remains unknown how NP-binding specificity to vRNA is established. Here we introduced nucleotide changes to vRNA to examine whether primary sequence can affect NP binding. Our findings demonstrate that NP binding is indeed susceptible to sequence alterations, as NP peaks can be lost or appear de novo at mutated sites. Unexpectedly, nucleotide changes not only affect NP binding locally at the site of mutation, but also impact NP binding at distal regions that have not been modified. Taken together, our results suggest that NP binding is not regulated by primary sequence alone, but that a network formed by multiple segments governs the deposition of NP on vRNA.

Proliferating cell nuclear antigen impairs the nuclear import of influenza A virus PB2 and suppresses virus replication.

The genome of Influenza A virus (IAV) transcribes and replicates in the nucleus of cells and the viral ribonucleoprotein (vRNP) complex plays an important role in viral replication. As a major component of the vRNP complex, the polymerase basic protein 2 (PB2) is translocated to the nucleus via its nuclear localization signals mediated by the importins. Herein, it was identified proliferating cell nuclear antigen (PCNA) as an inhibitor of nuclear import of PB2 and subsequent viral replication. Mechanically, PCNA interacted with PB2 and inhibited the nuclear import of PB2. Furthermore, PCNA decreased the binding efficiency of PB2 with importin alpha (importin α) and the K738, K752, and R755 of PB2 were identified as the key sites binding with PCNA and importin α. Furthermore, PCNA was demonstrated to retrain the vRNP assembly and polymerase activity. Taken together, the results demonstrated that PCNA impaired the nuclear import of PB2, vRNP assembly and polymerase activity, which negatively regulated virus replication.

High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing

The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

Results from a test-and-treat study for influenza among residents of homeless shelters in King County, WA: A stepped-wedge cluster-randomized trial.

Persons experiencing homelessness face increased risk of influenza as overcrowding in congregate shelters can facilitate influenza virus spread. Data regarding on-site influenza testing and antiviral treatment within homeless shelters remain limited. We conducted a cluster-randomized stepped-wedge trial of point-of-care molecular influenza testing coupled with antiviral treatment with baloxavir or oseltamivir in residents of 14 homeless shelters in Seattle, WA, USA. Residents ≥3 months with cough or ≥2 acute respiratory illness (ARI) symptoms and onset <7 days were eligible. In control periods, mid-nasal swabs were tested for influenza by reverse transcription polymerase chain reaction (RT-PCR). The intervention period included on-site rapid molecular influenza testing and antiviral treatment for influenza-positives if symptom onset was <48 h. The primary endpoint was monthly influenza virus infections in the control versus intervention periods. Influenza whole genome sequencing was performed to assess transmission and antiviral resistance. During 11/15/2019-4/30/2020 and 11/2/2020-4/30/2021, 1283 ARI encounters from 668 participants were observed. Influenza virus was detected in 51 (4%) specimens using RT-PCR (A = 14; B = 37); 21 influenza virus infections were detected from 269 (8%) intervention-eligible encounters by rapid molecular testing and received antiviral treatment. Thirty-seven percent of ARI-participant encounters reported symptom onset < 48 h. The intervention had no effect on influenza virus transmission (adjusted relative risk 1.73, 95% confidence interval [CI] 0.50-6.00). Of 23 influenza genomes, 86% of A(H1N1)pdm09 and 81% of B/Victoria sequences were closely related. Our findings suggest feasibility of influenza test-and-treat strategies in shelters. Additional studies would help discern an intervention effect during periods of increased influenza activity.

MicroRNA-9-1 Attenuates Influenza A Virus Replication via Targeting Tankyrase 1

An unstable influenza genome leads to the virus resistance to antiviral drugs that target viral proteins. Thus, identification of host factors essential for virus replication may pave the way to develop novel antiviral therapies. In this study, we investigated the roles of the poly(ADP-ribose) polymerase enzyme, tankyrase 1 (TNKS1), and the endogenous small noncoding RNA, miR-9-1, in influenza A virus (IAV) infection. Increased expression of TNKS1 was observed in IAV-infected human lung epithelial cells and mouse lungs. TNKS1 knockdown by RNA interference repressed influenza viral replication. A screen using TNKS1 3′-untranslation region (3′-UTR) reporter assays and predicted microRNAs identified that miR-9-1 targeted TNKS1. Overexpression of miR-9-1 reduced influenza viral replication in lung epithelial cells as measured by viral mRNA and protein levels as well as virus production. miR-9-1 induced type I interferon production and enhanced the phosphorylation of STAT1 in cell culture. The ectopic expression of miR-9-1 in the lungs of mice by using an adenoviral viral vector enhanced type I interferon response, inhibited viral replication, and reduced susceptibility to IAV infection. Our results indicate that miR-9-1 is an anti-influenza microRNA that targets TNKS1 and enhances cellular antiviral state.

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.

The influenza A virus genome packaging network - complex, flexible and yet unsolved.

The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA–RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA–RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA–RNA interactions. We speculate that identifying such functional intersegmental RNA–RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome.

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.