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Previous studies showed that ZAPL (PARP-13.1) exerts its antiviral activity via its N-terminal zinc fingers that bind the mRNAs of some viruses, leading to mRNA degradation. Here we identify a different antiviral activity of ZAPL that is directed against influenza A virus. This ZAPL antiviral activity involves its C-terminal PARP domain, which binds the viral PB2 and PA polymerase proteins, leading to their proteasomal degradation. After the PB2 and PA proteins are poly(ADP-ribosylated), they are associated with the region of ZAPL that includes both the PARP domain and the adjacent WWE domain that is known to bind poly(ADP-ribose) chains. These ZAPL-associated PB2 and PA proteins are then ubiquitinated, followed by proteasomal degradation. This antiviral activity is counteracted by the viral PB1 polymerase protein, which binds close to the PARP domain and causes PB2 and PA to dissociate from ZAPL and escape degradation, explaining why ZAPL only moderately inhibits influenza A virus replication. Hence influenza A virus has partially won the battle against this newly identified ZAPL antiviral activity. Eliminating PB1 binding to ZAPL would be expected to substantially increase the inhibition of influenza A virus replication, so that the PB1 interface with ZAPL is a potential target for antiviral development.

Influenza viruses annually kill 290,000–650,000 people worldwide. Antivirals can reduce death tolls. Baloxavir, the recently approved influenza antiviral, inhibits initiation of viral mRNA synthesis, whereas oseltamivir, an older drug, inhibits release of virus progeny. Baloxavir blocks virus replication more rapidly and completely than oseltamivir, reducing the duration of infectiousness. Hence, early baloxavir treatment may indirectly prevent transmission. Here, we estimate impacts of ramping up and accelerating baloxavir treatment on population-level incidence using a new model that links viral load dynamics from clinical trial data to between-host transmission. We estimate that ~22 million infections and >6,000 deaths would have been averted in the 2017–2018 epidemic season by administering baloxavir to 30% of infected cases within 48 h after symptom onset. Treatment within 24 h would almost double the impact. Consequently, scaling up early baloxavir treatment would substantially reduce influenza morbidity and mortality every year. The development of antivirals against the SARS-CoV2 virus that function like baloxavir might similarly curtail transmission and save lives. Here, the authors implement a mathematical model that describes how Baloxavir antiviral-induced inhibition of influenza virus replication in infected individuals affects the spread of the virus during epidemics, suggesting that both the scaling up and acceleration of treatment would avert substantial influenza morbidity and mortality every year.

Groovy The structure of the influenza A virus nucleoprotein has proved elusive. But now its crystal structure has been determined, to reveal a crescent-shaped molecule containing connected head and body regions with a groove in between to accommodate the viral RNA. Now the contact points between this protein and other parts of the virus particle can be visualized, which should help in the development of antiviral therapeutics for influenza. The structure of the influenza virus A nucleoprotein is visualized, revealing connected head and body regions with a groove in between for the viral RNA to sit. Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication 1 . Here we report a 3.2 Å crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein 2 , 3 . Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.

The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. The ubiquitin-like protein ISG15 can be covalently linked to cellular and viral proteins, but the consequences of this ‘ISGylation’ remain largely unknown. Here, Zhao et al. show that ISGylation of the influenza B virus nucleoprotein inhibits formation of a functional viral replication complex.

We evaluated the population-level benefits of expanding treatment with the antiviral drug Paxlovid (nirmatrelvir/ritonavir) in the United States for SARS-CoV-2 Omicron variant infections. Using a multiscale mathematical model, we found that treating 20% of symptomatic case-patients with Paxlovid over a period of 300 days beginning in January 2022 resulted in life and cost savings. In a low-transmission scenario (effective reproduction number of 1.2), this approach could avert 0.28 million (95% CI 0.03-0.59 million) hospitalizations and save US $56.95 billion (95% CI US $2.62-$122.63 billion). In a higher transmission scenario (effective reproduction number of 3), the benefits increase, potentially preventing 0.85 million (95% CI 0.36-1.38 million) hospitalizations and saving US $170.17 billion (95% CI US $60.49-$286.14 billion). Our findings suggest that timely and widespread use of Paxlovid could be an effective and economical approach to mitigate the effects of COVID-19.

The NS1 protein of influenza A virus (NS1A protein) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. Its N-terminal RNA-binding domain binds dsRNA. The only amino acid absolutely required for dsRNA binding is the R at position 38. To identify the role of this dsRNA-binding activity during influenza A virus infection, we generated a recombinant influenza A/Udorn/72 virus expressing an NS1A protein containing an RNA-binding domain in which R38 is mutated to A. This R38A mutant virus is highly attenuated, and the mutant NS1A protein, like the WT protein, is localized in the nucleus. Using the R38A mutant virus, we establish that dsRNA binding by the NS1A protein does not inhibit production of IFN-β mRNA. Rather, we demonstrate that the primary role of this dsRNA-binding activity is to protect the virus against the antiviral state induced by IFN-β. Pretreatment of A549 cells with IFN-β for 6 h did not inhibit replication of WT Udorn virus, whereas replication of R38A mutant virus was inhibited 1,000-fold. Using both RNA interference in A549 cells and mouse knockout cells, we show that this enhanced sensitivity to IFN-β-induced antiviral activity is due predominantly to the activation of RNase L. Because activation of RNase L is totally dependent on dsRNA activation of 2'-5' oligo (A) synthetase (OAS), it is likely that the primary role of dsRNA binding by the NS1A protein in virus-infected cells is to sequester dsRNA away from 2'-5' OAS.

The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the molecular biology of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance our understanding of the molecular mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the nonstructural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.

ISG15 is an IFN-α/β-induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a \"loss of function\" mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.

IFN-α/β plays an essential role in innate immunity against viral and bacterial infection. Among the proteins induced by IFN-α/β are the ubiquitin-like ISG15 protein and its E1- (Ube1L) and E2- (UbcH8) conjugating enzymes, leading to the conjugation of ISG15 to cellular proteins. It is likely that ISG15 conjugation plays an important role in antiviral response because a human virus, influenza B virus, inhibits ISG15 conjugation. However, the biological function of ISG15 modification remains unknown, largely because only a few human ISG15 target proteins have been identified. Here we purify ISG15-modified proteins from IFN-β-treated human (HeLa) cells by using double-affinity selection and use mass spectroscopy to identify a large number (158) of ISG15 target proteins. Eight of these proteins were subjected to further analysis and verified to be ISG15 modified in IFN-β-treated cells, increasing the likelihood that most, if not all, targets identified by mass spectroscopy are bona fide ISG15 targets. Several of the targets are IFN-α/β-induced antiviral proteins, including PKR, MxA, HuP56, and RIG-I, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling/polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions. By targeting a wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-α/β.