Cellular protein interaction networks are integral to host defence and immune signalling pathways, which are often hijacked by viruses via protein interactions. However, the comparative virus–host protein interaction networks and how these networks control host immunity and viral infection remain to be elucidated. Here, we mapped protein interactomes between human host and several influenza A viruses (IAV). Comparative analyses of the interactomes identified common and unique interaction patterns regulating innate immunity and viral infection. Functional screening of the ‘core‘ interactome consisting of common interactions identified five novel host factors regulating viral infection. Plakophilin 2 (PKP2), an influenza PB1-interacting protein, restricts IAV replication and competes with PB2 for PB1 binding. The binding competition leads to perturbation of the IAV polymerase complex, thereby limiting polymerase activity and subsequent viral replication. Taken together, comparative analyses of the influenza–host protein interactomes identified PKP2 as a natural inhibitor of IAV polymerase complex.

Influenza A virus (IAV) is a highly transmissible respiratory pathogen and a major cause of morbidity and mortality around the world. Nucleoprotein (NP) is an abundant IAV protein essential for multiple steps of the viral life cycle. Our recent proteomic study of the IAV-host interaction network found that TRIM41 (tripartite motif-containing 41), a ubiquitin E3 ligase, interacted with NP. However, the role of TRIM41 in IAV infection is unknown. Here, we report that TRIM41 interacts with NP through its SPRY domain. Furthermore, TRIM41 is constitutively expressed in lung epithelial cells, and overexpression of TRIM41 inhibits IAV infection. Conversely, RNA interference (RNAi) and knockout of TRIM41 increase host susceptibility to IAV infection. As a ubiquitin E3 ligase, TRIM41 ubiquitinates NP and in cells. The TRIM41 mutant lacking E3 ligase activity fails to inhibit IAV infection, suggesting that the E3 ligase activity is indispensable for TRIM41 antiviral function. Mechanistic analysis further revealed that the polyubiquitination leads to NP protein degradation and viral inhibition. Taking these observations together, TRIM41 is a constitutively expressed intrinsic IAV restriction factor that targets NP for ubiquitination and protein degradation. Influenza control strategies rely on annual immunization and require frequent updates of the vaccine, which is not always a foolproof process. Furthermore, the current antivirals are also losing effectiveness as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new antiviral mechanisms and develop therapeutic drugs based on these mechanisms. Targeting the virus-host interface is an emerging new strategy because host factors controlling viral replication activity will be ideal candidates, and cellular proteins are less likely to mutate under drug-mediated selective pressure. Here, we show that the ubiquitin E3 ligase TRIM41 is an intrinsic host restriction factor to IAV. TRIM41 directly binds the viral nucleoprotein and targets it for ubiquitination and proteasomal degradation, thereby limiting viral infection. Exploitation of this natural defense pathway may open new avenues to develop antiviral drugs targeting the influenza virus.

No abstract

Tripartite motif (TRIM) proteins have been found in a variety of physiological processes; however, the role of TRIM proteins in host defense to viral infection is emerging in recent years. TRIM proteins have been shown to restrict viruses at various stages of viral life cycle through common and distinct mechanisms. TRIM proteins restrict viral infection by directly interacting with viral proteins. Furthermore, TRIM proteins regulate innate immunity and adaptive immunity to impede viral infection. To subvert host defense, viruses also evolve a new evasion strategy by targeting TRIM proteins. In this review, we highlight recent advances which deepen our understanding of the role of TRIM proteins in host defense and the diverse antiviral mechanisms of TRIM proteins.

NF-κB signaling regulates diverse processes such as cell death, inflammation, immunity, and cancer. The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin, which is assembled by the linear ubiquitin chain assembly complex (LUBAC) and the ubiquitin-conjugating enzyme UBE2L3. Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain, thus inhibiting NF-κB signaling. Despite the essential role of OTULIN in NF-κB signaling has been established, the regulatory mechanism for OTULIN is not well elucidated. To discover the potential regulators of OTULIN, we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins, including LUBAC and tripartite motif-containing protein 32 (TRIM32). TRIM32 is known to activate NF-κB signaling, but the mechanism is not clear. Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN. Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation. Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN, thereby activating NF-κB signaling. Taken together, we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.

Vesicular stomatitis virus (VSV) is a zoonotic, negative-stranded RNA virus of the family Rhabdoviridae. The nucleoprotein (N) of VSV protects the viral genomic RNA and plays an essential role in viral transcription and replication, which makes the nucleoprotein an ideal target of host defense. However, whether and how host innate/intrinsic immunity limits VSV infection by targeting the N protein are unknown. In this study, we found that the N protein of VSV (VSV-N) interacted with a ubiquitin E3 ligase, tripartite motif protein 41 (TRIM41). Overexpression of TRIM41 inhibited VSV infection. Conversely, the depletion of TRIM41 increased host susceptibility to VSV. Furthermore, the E3 ligase defective mutant of TRIM41 failed to limit VSV infection, suggesting the requirement of the E3 ligase activity of TRIM41 in viral restriction. Indeed, TRIM41 ubiquitinated VSV-N in cells and in vitro. TRIM41-mediated ubiquitination leads to the degradation of VSV-N through proteasome, thereby limiting VSV infection. Taken together, our study identifies TRIM41 as a new intrinsic immune factor against VSV by targeting the viral nucleoprotein for ubiquitination and subsequent protein degradation.

The global outbreak and rapid spread of SARS-CoV-2 created an urgent need for large scale testing of populations. There is a demand for high throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled-PCR protocol for testing nasopharyngeal swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. Six-hundred and thirty (630) samples were used in this study. Individual positive samples with Ct values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5) and individual positive samples with Ct values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

Genomic sequencing has played a major role in understanding the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the current pandemic, it is essential that SARS-CoV-2 viruses are sequenced regularly to determine mutations and genomic modifications in different geographical locations. In this study we sequenced SARS-CoV-2 from 5 clinical samples obtained in Oklahoma, USA during different time points of pandemic presence in the state. One sample from the initial days of the pandemic in the state and 4 during the peak in Oklahoma were sequenced. Previously reported mutations including D614G in S gene, P4715L in ORF1ab, S194L, R203K and G204R in N gene were identified in the genomes sequenced in this study. Possible novel mutations were also detected such as G1167V in S gene, A6269S and P3371S in ORF1ab, T28I in ORF7b, G96R in ORF8. Phylogenetic analysis of the genomes showed similarity to viruses from across the globe. These novel mutations and phylogenetic analysis emphasize the contagious nature of the virus.