Summary Lassa fever virus (LASV) causes thousands of deaths yearly and is a biological threat agent, for which there is no vaccine and limited therapy1. The nucleoprotein (NP) of LASV plays essential roles in viral RNA synthesis and immune suppression2-6, the molecular mechanisms of which are poorly understood. Here, we report the crystal structure of LASV NP at 1.80 Angstrom resolution, which reveals N- and C-domains with structures unlike any of the reported viral NPs7-10. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3′-5′ exoribonuclease activity involved in suppressing interferon induction. This is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and suggests unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. Here we show that KSHV RTA interacts with the RBP-J protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-J recognition sites on DNA and results in the replacement of RBP-J 's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-J −/− mice, a defect that can be reversed by expression of RBP-J . In vitro, RTA binds to two adjacent regions of RBP-J , one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency.

Difficulties in efficiently propagating

The NF-B signaling pathway has previously been shown to be required for efficient influenza A virus replication, although the molecular mechanism is not well understood. In this study, we identified a specific step of the influenza virus life cycle that is influenced by NF-B signaling by using two known NF-B inhibitors and a variety of influenza virus-specific assays. The results of time course experiments suggest that the NF-B inhibitors Bay11-7082 and ammonium pyrrolidinedithiocarbamate inhibited an early postentry step of viral infection, but they did not appear to affect the nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. Instead, we found that the levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by the inhibitors in virus-infected cells, indicating that NF-B signaling is intimately involved in the vRNA synthesis. Furthermore, we showed that the NF-B inhibitors specifically diminished influenza virus RNA transcription from the cRNA promoter but not from the vRNA promoter in a reporter assay, a result which is consistent with data obtained from virus-infected cells. The overexpression of the p65 NF-B molecule could not only eliminate the inhibition but also activate influenza virus RNA transcription from the cRNA promoter. Finally, using p65-specific small interfering RNA, we have shown that p65 knockdown reduced the levels of influenza virus replication and vRNA synthesis. In summary, we have provided evidence showing, for the first time, that the NF-B host signaling pathway can differentially regulate influenza virus RNA synthesis, which may also offer some new perspectives into understanding the host regulation of RNA synthesis by other RNA viruses.

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5 and 3 untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5 and 66 bases of 3 coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5 coding region but is independent of coding sequences at the 3 end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5 and 3 ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.The genome of influenza A virus consists of eight negativesense RNA segments (called vRNAs) that together encode the 11 known viral proteins (2, 14, 17). During its assembly, a nascent influenza virus particle must incorporate (package) at least one copy of each vRNA in order to become infectious. The mechanisms that govern influenza virus RNA packaging are poorly understood. Although the virus packages its genomic segments selectively in preference to most cellular RNAs, it is not clear whether it discriminates among individual segments or packages them at random, and the molecular attributes of the vRNAs that allow them to be targeted into virions remain largely unknown.Each vRNA consists predominantly of coding sequences (in antisense orientation), flanked at both ends by untranslated regions (UTRs) that range from 19 to 58 bases long. Within these UTRs, the distal 12 and 13 noncoding bases that form the extreme 3Ј and 5Ј termini, respectively, of every segment are highly conserved among viral strains and among the eight segments themselves (4, 20). These distal conserved sequences are partially complementary to each other and so can anneal to form a bulged duplex structure (1, 7, 9, 13) that is essential for transcription and replication of the segment (3,8,15,18,19). The UTRs are believed to harbor cis-acting signals that contribute to RNA packaging, since the attachment of authentic UTRs onto a heterologous RNA can enable it to be packaged into, and transduced by, influenza virus particles (16). Packaging mediated solely by the UTRs is inefficient, however, and it has been difficult to distinguish the sequences responsible for packaging from those needed for other critical aspects of viral gene expression...

Background: Arenaviral nucleoproteins play a critical role in innate immune suppression.Results: Structures of Lassa nucleoprotein in complex with triphosphate dsRNA and Tacaribe virus nucleoprotein have been determined.Conclusion: Both Lassa and Tacaribe nucleoproteins can strongly inhibit IFN-β production by degrading immune-stimulatory dsRNA.Significance: A unique immune suppression mode of arenaviral nucleoproteins has been revealed.

Despite the success of vaccination to greatly mitigate or eliminate threat of diseases caused by pathogens, there are still known diseases and emerging pathogens for which the development of successful vaccines against them is inherently difficult. In addition, vaccine development for people with compromised immunity and other pre-existing medical conditions has remained a major challenge. Besides the traditional inactivated or live attenuated, virus-vectored and subunit vaccines, emerging non-viral vaccine technologies, such as viral-like particle and nanoparticle vaccines, DNA/RNA vaccines, and rational vaccine design, offer innovative approaches to address existing challenges of vaccine development. They have also significantly advanced our understanding of vaccine immunology and can guide future vaccine development for many diseases, including rapidly emerging infectious diseases, such as COVID-19, and diseases that have not traditionally been addressed by vaccination, such as cancers and substance abuse. This review provides an integrative discussion of new non-viral vaccine development technologies and their use to address the most fundamental and ongoing challenges of vaccine development.

Host signaling pathways play important roles in the replication of influenza virus, but their functional effects remain to be characterized at the molecular level. Here we identify two receptor tyrosine kinase inhibitors (RTKIs) of the tyrphostin class that exhibit robust antiviral activity against influenza A virus replication in cultured cells. One of these (AG879) is a selective inhibitor of the nerve growth factor receptor and human epidermal growth factor receptor 2 (TrkA/HER2) signaling; the other, tyrphostin A9 (A9), inhibits the plateletderived growth factor receptor (PDGFR) pathway. We find that each inhibits at least three postentry steps of the influenza virus life cycle: AG879 and A9 both strongly inhibit the synthesis of all three influenza virus RNA species, block Crm1-dependent nuclear export, and also prevent the release of viral particles through a pathway that is modulated by the lipid biosynthesis enzyme farnesyl diphosphate synthase (FPPS). Tests of short hairpin RNA (shRNA) knockdown and additional small-molecule inhibitors confirmed that interventions targeting TrkA can suppress influenza virus replication. Our study suggests that host cell receptor tyrosine kinase signaling is required for maximal influenza virus RNA synthesis, viral ribonucleoprotein (vRNP) nuclear export, and virus release and that specific RTKIs hold promise as novel anti-influenza virus therapeutics.