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Single-cycle infectious virus can elicit close-to-natural immune response and memory. One approach to generate single-cycle severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN-infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single-cycle vaccines for SARS-CoV-2.

Infection with seasonal as well as highly pathogenic avian influenza A virus (IAV) causes significant morbidity and mortality worldwide. As a major virulence factor, PB1-F2 protein of IAV affects the severity of disease through multiple mechanisms including perturbation of host innate immune response. Macrophages are known to phagocytose extracellular PB1-F2 protein aggregate, leading to hyperactivation of NLRP3 inflammasome and excessive production of IL-1β and IL-18. On the other hand, when expressed intracellularly PB1-F2 suppresses NLRP3 inflammasome maturation. How extracellular and intracellular PB1-F2 orchestrates to drive viral pathogenesis remains unclear. In this study, we demonstrated the suppression of NLRP3 inflammasome activation and IL-1β secretion by PB1-F2 of highly pathogenic influenza A (H7N9) virus in infected human monocyte-derived macrophages. Mechanistically, H7N9 PB1-F2 selectively mitigated RNA-induced NLRP3 inflammasome activation by inhibiting the interaction between NLRP3 and MAVS. Intracellular PB1-F2 of H7N9 virus did not affect extracellular PB1-F2-induced NLRP3 inflammasome maturation. In contrast, PB1-F2 of WSN laboratory strain of human IAV effectively suppressed IL-1β processing and secretion induced by various stimuli including NLRP3, AIM2, and pro-IL-1β. This subtype-specific effect of PB1-F2 on inflammasome activation correlates with the induction of a proinflammatory cytokine storm by H7N9 but not WSN virus. Our findings on selective suppression of MAVS-dependent activation of NLRP3 inflammasome by H7N9 PB1-F2 have implications in viral pathogenesis and antiviral development.

Influenza A virus (IAV) causes not only seasonal respiratory illness, but also outbreaks of more severe disease and pandemics when novel strains emerge as a result of reassortment or interspecies transmission. PB1-F2 is an IAV protein expressed from the second open reading frame of PB1 gene. Small as it is, PB1-F2 is a critical virulence factor. Multiple key amino acid residues and motifs of PB1-F2 have been shown to influence the virulence of IAV in a strain- and host-specific manner, plausibly through the induction of apoptotic cell death, modulation of type I IFN response, activation of inflammasome, and facilitation of secondary bacterial infection. However, the exact role of PB1-F2 in IAV pathogenesis remains unexplained. Through reanalysis of the current literature, we redefine PB1-F2 as an ambivalent innate immune modulator that determines IAV infection outcome through induction of immune cell death, differential modulation of early- and late-type I IFN response, and promotion of pathogenic inflammation. PB1-F2 functions both intracellularly and extracellularly. Further investigations of the mechanistic details of PB1-F2 action will shed new light on immunopathogenesis of IAV infection.

Rapid development and successful use of vaccines against SARS-CoV-2 might hold the key to curb the ongoing pandemic of COVID-19. Emergence of vaccine-evasive SARS-CoV-2 variants of concern (VOCs) has posed a new challenge to vaccine design and development. One urgent need is to determine what types of variant-specific and bivalent vaccines should be developed. Here, we compared homotypic and heterotypic protection against SARS-CoV-2 infection of hamsters with monovalent and bivalent whole-virion inactivated vaccines derived from representative VOCs. In addition to the ancestral SARS-CoV-2 Wuhan strain, Delta (B.1.617.2; δ) and Theta (P.3; θ) variants were used in vaccine preparation. Additional VOCs including Omicron (B.1.1.529) and Alpha (B.1.1.7) variants were employed in the challenge experiment. Consistent with previous findings, Omicron variant exhibited the highest degree of immune evasion, rendering all different forms of inactivated vaccines substantially less efficacious. Notably, monovalent and bivalent Delta variant-specific inactivated vaccines provided optimal protection against challenge with Delta variant. Yet, some cross-variant protection against Omicron and Alpha variants was seen with all monovalent and bivalent inactivated vaccines tested. Taken together, our findings support the notion that an optimal next-generation inactivated vaccine against SARS-CoV-2 should contain the predominant VOC in circulation. Further investigations are underway to test whether a bivalent vaccine for Delta and Omicron variants can serve this purpose.

Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon β in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus., Author summary Exactly why avian influenza A (H5N1) and (H7N9) viruses cause severe diseases in humans remains unclear. PB1-F2 protein encoded by influenza A virus is one virulence factor that might make a difference. In this study we show that PB1-F2 protein of H7N9 virus is particularly strong in the suppression of host antiviral defense. This was achieved by inhibiting a key protein in cell signaling named MAVS. PB1-F2 directs MAVS for degradation and prevents MAVS from forming protein aggregates required for full activation. A recombinant virus in which PB1-F2 of H7N9 has been deleted can activate host antiviral response robustly. Our findings reveal a novel mechanism by which PB1-F2 protein of H7N9 virus prevents MAVS aggregation and promotes MAVS degradation, leading to the suppression of host antiviral defense.

PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-β by the IAV RNP complex was most robust when both RIG-I and PACT were expressed. PACT-dependent activation of IFN-β production was suppressed by the IAV polymerase subunits, polymerase acidic protein, polymerase basic protein 1 (PB1), and PB2. PACT associated with PA, PB1, and PB2. Compromising PACT in IAV-infected A549 cells resulted in the augmentation of viral RNA (vRNA) transcription and replication and IFN-β production. Furthermore, vRNA replication was boosted by knockdown of PACT in both A549 cells and IFN-deficient Vero cells. Thus, the antiviral activity of PACT is mediated primarily via its interaction with and inhibition of IAV polymerase. Taken together, our findings reveal a new facet of the host-IAV interaction in which the interplay between PACT and IAV polymerase affects the outcome of viral infection and antiviral response.-Chan, C.-P., Yuen, C.-K., Cheung, P.-H. H., Fung, S.-Y., Lui, P.-Y., Chen, H., Kok, K.-H., Jin, D.-Y. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

To announce or not to announce: What is known about the 2016–2017 influenza season in Hong Kong?