Significance: To successfully establish infection, viral pathogens have to overcome the interferon (IFN)-mediated antiviral response. Previous studies revealed that the viral accessory protein Orf6 of SARS-CoV and SARS-CoV-2 is able to inhibit STAT1 nuclear translocation to block IFN signaling. In this study, we report that Orf6 localizes at the nuclear pore complex (NPC) where it binds directly to the Nup98-Rae1 complex to target the nuclear import pathway and mediate this inhibition. A better understanding of the strategies used by viruses to subvert host immune responses is critical for the design of novel antivirals and vaccines.

SARS-CoV-2 Nsp1 protein binds the mRNA export receptor NXF1-NXT1 to inhibit nuclear export of mRNAs and promote infection.

Aa: amino acid; ATF6: activating transcription factor 6; ATG5: autophagy related 5; CCPG1: cell cycle progression 1; CFTR: CF transmembrane conductance regulator; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CQ: chloroquine; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HSV-1: herpes simplex virus type 1; IFIT1: interferon induced protein with tetratricopeptide repeats 1; IFNB1/IFN-β: interferon beta 1; IRF3: interferon regulatory factor 3; ISG15: ISG15 ubiquitin like modifier; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NFKB/NF-κB: nuclear factor kappa B; NSP6: non-structural protein 6; Δ106-108: deletion of amino acids 106-108 in NSP6 of SARS-CoV-2; Δ105-107: deletion of amino acids 105-107 in NSP6 of SARS-CoV-2; RETREG1/FAM134B: reticulophagy regulator 1; RIGI/DDX58: RNA sensor RIG-I; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1.

Gene expression in eukaryotes begins with transcription in the nucleus, followed by the synthesis of messenger RNA (mRNA), which is then exported to the cytoplasm for its translation into proteins. Along with transcription and translation, mRNA export through the nuclear pore complex (NPC) is an essential regulatory step in eukaryotic gene expression. Multiple factors regulate mRNA export and hence gene expression. Interestingly, proteins from certain types of viruses interact with these factors in infected cells, and such an interaction interferes with the mRNA export of the host cell in favor of viral RNA export. Thus, these viruses hijack the host mRNA nuclear export mechanism, leading to a reduction in host gene expression and the downregulation of immune/antiviral responses. On the other hand, the viral mRNAs successfully evade the host surveillance system and are efficiently exported from the nucleus to the cytoplasm for translation, which enables the continuation of the virus life cycle. Here, we present this review to summarize the mechanisms by which viruses suppress host mRNA nuclear export during infection, as well as the key strategies that viruses use to facilitate their mRNA nuclear export. These studies have revealed new potential antivirals that may be used to inhibit viral mRNA transport and enhance host mRNA nuclear export, thereby promoting host gene expression and immune responses.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus. Influenza virus remains a major public health threat killing ~250,000–500,000 people yearly. In pandemic years, influenza infection can lead to even higher mortality rates, as in 1918 when at least 20 million deaths occurred worldwide. Available treatments include vaccines and a few antiviral drugs, but both are limited by mutability of the virus and the development of resistance. Here we leveraged on knowledge of our previous work that uncovered a unique influenza virus pathway inside the nucleus of the host cell, which have the potential to be targeted by a novel therapeutic strategy. We designed and performed a new screen to identify chemical compounds that inhibited viral mRNA production and transport from the nucleus to the cytoplasm of the host cell. Among these compounds, we highlight a novel nuclear export inhibitor of a subset of viral and cellular mRNAs that does not substantially alter bulk cellular RNA export or levels at non-toxic dosages. Consequently, this small molecule inhibits virus replication and therefore can be used as a lead for drug development and serve as an experimental tool for further understanding key steps of the influenza virus mRNA nuclear export pathway.

• Scientific questions: Ongoing viral transmission and the increasing threat of drug resistance highlight the pressing need for the development of new anti-monkeypox virus therapies. The roles of microribonucleic acids (miRNAs), which regulate gene expression at the post-transcriptional level, in poxvirus infection remain poorly understood and require further investigation. • Evidence before this study: Increasing evidence has shown that miRNAs are key regulators of gene expression. A growing number of miRNAs have been implicated in various pathological conditions, including viral infections. Previous studies reported that miR-24-3p inhibits the replication of vesicular stomatitis virus (VSV), hepatitis C virus (HCV), H5N1 influenza A virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). • New findings: miR-24-3p expression is significantly reduced during vaccinia virus (VACV) infection, whereas enforced overexpression markedly inhibits VACV replication, identifying miR-24-3p as a functional antiviral miRNA in poxvirus infection. KIF21B is identified as a promoter of VACV replication, uncovering a novel miRNA-host factor axis (miR-24-3p-KIF21B) that regulates VACV replication. Lipid nanoparticle-mediated delivery of miR-24-3p confers significant protection in a VACV-infected mouse model, providing in vivo evidence for miRNA-based therapy against poxvirus infection. • Significance of the study: These findings broaden our knowledge of the interplay between miRNAs and VACV, fill a critical knowledge gap in poxvirus biology, and highlight miR-24-3p as a promising therapeutic candidate for the prevention and treatment of orthopoxvirus infections.