H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.

Host factors play important roles in influenza A virus (IAV) replication. In order to identify novel host factors involved in IAV replication, we compared the differentially expressed genes in A549 cells after IAV infection. We found that lncRNA lnc-RPS6P3 was up-regulated upon viral infection and poly(I:C) and IFN-β treatment, indicating it was an interferon-stimulated gene. Functional analysis demonstrated that overexpression of lnc-RPS6P3 inhibited IAV replication while knockdown of lnc-RPS6P3 promoted viral infection in A549 cells. Lnc-RPS6P3 inhibited both transcription and replication of IAV. Further study showed that lnc-RPS6P3 interacted with viral NP and interfered with NP self-oligomerization and, consequently, inhibited vRNP activity. In addition, lnc-RPS6P3 interacted with viral NS1 and reduced the interaction of NS1 and RIG-I; it also attenuated the inhibitory effect of NS1 on IFN-β stimulation. In conclusion, we revealed that lnc-RPS6P3 is an interferon-stimulated gene that inhibits IAV replication and attenuates the inhibitory effect of NS1 on innate immune response.

Influenza A virus (IAV) relies on intricate and highly coordinated associations with host factors for efficient replication and transmission. Characterization of such factors holds great significance for development of anti-IAV drugs. Our study identified protein arginine methyltransferase 5 (PRMT5) as a novel host factor indispensable for IAV replication. Silencing PRMT5 resulted in drastic repression of IAV replication. Our findings revealed that PRMT5 interacts with each protein component of viral ribonucleoproteins (vRNPs) and promotes arginine symmetric dimethylation of polymerase basic 2 (PB2). Overexpression of PRMT5 enhanced viral polymerase activity in a dose-dependent manner, emphasizing its role in genome transcription and replication of IAV. Moreover, analysis of PB2 protein sequences across various subtypes of IAVs demonstrated the high conservation of potential RG motifs recognized by PRMT5. Overall, our study suggests that PRMT5 supports IAV replication by facilitating viral polymerase activity by interacting with PB2 and promoting its arginine symmetric dimethylation. This study deepens our understanding of how IAV manipulates host factors to facilitate its replication and highlights the great potential of PRMT5 to serve as an anti-IAV therapeutic target.

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.

Influenza A virus (IAV) RNA-dependent RNA polymerase (vPol) is a heterotrimer composed of PB2, PB1, and PA, which, together with vRNA and nucleoprotein (NP), forms viral ribonucleoprotein (vRNP) complex to direct the transcription and replication of the viral genome. Host factor ANP32 proteins have been proved to be associated with vRNP and are essential for polymerase activity and cross-species restriction of avian influenza virus. However, the molecular mechanism by which ANP32 supports polymerase activity is largely unknown. Here, we identified that KPNA6 is associated with ANP32A/B and vRNP of the influenza virus. Both knockout and overexpression of KPNA6 downregulate the replication of the influenza virus by inhibiting the polymerase activity, indicating that a certain level of KPNA6 is beneficial for efficient replication of the influenza virus. Furthermore, we demonstrate that overexpression of KPNA6 or its nuclear importing domain negative mutation inhibited the interaction between ANP32 and vRNP, thus reducing the polymerase activity. Our results revealed the role of KPNA6 in interacting with both ANP32A/B and vRNP to maintain viral polymerase activity and provided new insights for further understanding of the mechanism by which ANP32 supports influenza polymerase. IMPORTANCE Host factor ANP32 plays a fundamental role in supporting the polymerase activity of influenza viruses, but the underlying mechanism is largely unknown. Here, we propose that KPNA6 is involved in the function of ANP32A/B to support influenza virus polymerase by interacting with both vRNP and ANP32A/B. The proper amount of KPNA6 and ANP32 proteins in the KPNA6-ANP32-vRNP complex is crucial for maintaining the viral polymerase activity. The KPNA6 may contribute to maintaining stable interaction between vRNA and ANP32 proteins in the nucleus, and this function is independent of the known importing domain of KPNA6. Our research reveals a role of KNPA6 associated with ANP32 proteins that support the viral polymerase and suggests a new perspective for developing antiviral strategies.

The genome of influenza A virus (IAV) comprises eight pinlike genomic segments called vRNPs enclosed in viral capsid. During infection, uncoating is the key step for viral replication and represents an antiviral therapeutic target, but it is difficult to observe the transient and dynamic event in detail. Here, we report a protocol for production of quantum dots-containing influenza virus particles by encapsulating quantum dot-conjugated vRNPs during viral assembly. These labeled virions can be used for monitoring viral trafficking in real time and studying viral uncoating processes.

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA-RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

The viral ribonucleoprotein (vRNP) of the influenza A virus (IAV) is responsible for the viral RNA transcription and replication in the nucleus, and its functions rely on host factors. Previous studies have indicated that eukaryotic translation elongation factor 1 delta (eEF1D) may associate with RNP subunits, but its roles in IAV replication are unclear. Herein, we showed that eEF1D was an inhibitor of IAV replication because knockout of eEF1D resulted in a significant increase in virus yield. eEF1D interacted with RNP subunits polymerase acidic protein (PA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), and also with nucleoprotein (NP) in an RNA-dependent manner. Further studies revealed that eEF1D impeded the nuclear import of NP and PA-PB1 heterodimer of IAV, thereby suppressing the vRNP assembly, viral polymerase activity, and viral RNA synthesis. Together, our studies demonstrate eEF1D negatively regulating the IAV replication by inhibition of the nuclear import of RNP subunits, which not only uncovers a novel role of eEF1D in IAV replication but also provides new insights into the mechanisms of nuclear import of vRNP proteins.IMPORTANCE Influenza A virus is the major cause of influenza, a respiratory disease in humans and animals. Different from most other RNA viruses, the transcription and replication of IAV occur in the cell nucleus. Therefore, the vRNPs must be imported into the nucleus for viral transcription and replication, which requires participation of host proteins. However, the mechanisms of the IAV-host interactions involved in nuclear import remain poorly understood. Here, we identified eEF1D as a novel inhibitor for the influenza virus life cycle. Importantly, eEF1D impaired the interaction between NP and importin α5 and the interaction between PB1 and RanBP5, which impeded the nuclear import of vRNP. Our studies not only reveal the molecular mechanisms of the nuclear import of IAV vRNP but also provide potential anti-influenza targets for antiviral development.

Influenza viruses are highly infectious and are the leading cause of human respiratory diseases and may trigger severe epidemics and occasional pandemics. Although antiviral drugs against influenza viruses have been developed, there is an urgent need to design new strategies to develop influenza virus inhibitors due to the increasing resistance of viruses toward currently available drugs. In this study, we examined the antiviral activity of natural compounds against the following influenza virus strains: A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and B/Lee/40. Papaverine (a nonnarcotic alkaloid that has been used for the treatment of heart disease, impotency, and psychosis) was found to be an effective inhibitor of multiple strains of influenza virus. Kinetic studies demonstrated that papaverine inhibited influenza virus infection at a late stage in the virus life cycle. An alteration in influenza virus morphology and viral ribonucleoprotein (vRNP) localization was observed as an effect of papaverine treatment. Papaverine is a well-known phosphodiesterase inhibitor and also modifies the mitogen-activated protein kinase (MAPK) pathway by downregulating the phosphorylation of MEK and extracellular signal-regulated kinase (ERK). Thus, the modulation of host cell signaling pathways by papaverine may be associated with the nuclear retention of vRNPs and the reduction of influenza virus titers. Interestingly, papaverine also inhibited paramyxoviruses parainfluenza virus 5 (PIV5), human parainfluenza virus 3 (HPIV3), and respiratory syncytial virus (RSV) infections. We propose that papaverine can be a potential candidate to be used as an antiviral agent against a broad range of influenza viruses and paramyxoviruses.IMPORTANCE Influenza viruses are important human pathogens that are the causative agents of epidemics and pandemics. Despite the availability of an annual vaccine, a large number of cases occur every year globally. Here, we report that papaverine, a vasodilator, shows inhibitory action against various strains of influenza virus as well as the paramyxoviruses PIV5, HPIV3, and RSV. A significant effect of papaverine on the influenza virus morphology was observed. Papaverine treatment of influenza-virus-infected cells resulted in the inhibition of virus at a later time in the virus life cycle through the suppression of nuclear export of vRNP and also interfered with the host cellular cAMP and MEK/ERK cascade pathways. This study explores the use of papaverine as an effective inhibitor of both influenza viruses as well as paramyxoviruses.

The role of microRNA (miRNA) in influenza A virus (IAV) host species specificity is not well understood as yet. Here, we show that a host miRNA, miR-1290, is induced through the extracellular signal-regulated kinase (ERK) pathway upon IAV infection and is associated with increased viral titers in human cells and ferret animal models. miR-1290 was observed to target and reduce expression of the host vimentin gene. Vimentin binds with the PB2 subunit of influenza A virus ribonucleoprotein (vRNP), and knockdown of vimentin expression significantly increased vRNP nuclear retention and viral polymerase activity. Interestingly, miR-1290 was not detected in either chicken cells or mouse animal models, and the 3\' UTR of the chicken vimentin gene contains no binding site for miR-1290. These findings point to a host species-specific mechanism by which IAV upregulates miR-1290 to disrupt vimentin expression and retain vRNP in the nucleus, thereby enhancing viral polymerase activity and viral replication.