Multiple interplays between viral and host factors are involved in influenza virus replication and pathogenesis. Several small RNAs have recently emerged as important regulators of host response to viral infections. The aim of this study was to characterize the functional role of hsa-miR-1975, a Y5 RNA-derived small RNA, in defending influenza virus and delineate the mechanisms. We performed high throughput sequencing of small RNAs in influenza virus-infected cells to identify up- or down- regulated small RNA species. The expression of the most abundant RNA species (hsa-miR-1975) was validated by stem-loop reverse transcription-polymerase chain reaction (RT-PCR). Antiviral effects of hsa-miR-1975 were confirmed by Western Blot, RT-PCR and plaque assay. In vitro perturbation of hsa-miR-1975 combined with exosomes isolation was used to elucidate the role and mechanism of hsa-miR-1975 in the context of antiviral immunity. Small RNA sequencing revealed that hsa-miR-1975 was the most up-regulated small RNA in influenza virus-infected cells. The amount of intracellular hsa-miR-1975 increased in the late stage of the influenza virus replication cycle. The increased hsa-miR-1975 was at least partially derived from degradation of Y5RNA as a result of cellular apoptosis. Unexpectedly, hsa-miR-1975 mimics inhibited influenza virus replication while hsa-miR-1975 sponges enhanced the virus replication. Moreover, hsa-miR-1975 was secreted in exosomes and taken up by the neighboring cells to induce interferon expression. Our findings unravel a critical role of Y-class small RNA in host’s defense against influenza virus infection and reveal its antiviral mechanism through exosome delivery. This may provide a new candidate for targeting influenza virus.

Recent Zika virus (ZIKV) epidemics necessitate the urgent development of effective drugs and vaccines, which can be accelerated by an enhanced understanding of ZIKV biology. One of the ZIKV structural proteins, precursor membrane (prM), plays an important role in the assembly of mature virions through cleavage of prM into M protein. Recent studies have suggested that prM protein might be implicated in the neurovirulence of ZIKV. Most vaccines targeting ZIKV include prM as the immunogen. Here, we review progress in our understanding of ZIKV prM protein and its application in ZIKV vaccine development.

Virus replication is mediated by interactions between the virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in the production of infectious virus particles. M2-K78R mutant progeny contained more hemagglutinin (HA) proteins, less viral RNAs, and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination by electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with the M1 protein to a lesser extent than for the wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal levels of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that the M2-K78R mutant virus induced autophagy and apoptosis earlier than did the wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating the efficient packaging of the viral genome into virus particles and the timing of virus-induced cell death. IMPORTANCE Annual epidemics and recurring pandemics of influenza viruses represent very high global health and economic burdens. The influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in virus entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N terminus of M2 proteins. However, viruses that are resistant to rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.

The main protease (M^pro) plays a crucial role in coronavirus, as it cleaves viral polyproteins and host cellular proteins to ensure successful replication. In this review, we discuss the preference in the recognition sequence of M^pro based on sequence-based studies and structural information and highlight the recent advances in computational and experimental approaches that have aided in discovering novel M^pro substrates. In addition, we provide an overview of the current understanding of M^pro host substrates and their implications for viral replication and pathogenesis. As M^pro has emerged as a promising target for the development of antiviral drugs, further insight into its substrate specificity may contribute to the design of specific inhibitors.

Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies. To date, several genome-wide RNAi screens have been successfully performed to identify host factors essential for influenza virus replication. However, the host factors identified by different research groups are not always consistent. Taking influenza virus screens as an example, we found that a number of screening parameters may directly or indirectly influence the primary hits identified by the screens. This review highlights the differences among the published genome-wide screening approaches and offers recommendations for performing a good pooled shRNA screen/selection.