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.

Neurologic dysfunctions such as Guillain-Barre' syndrome, encephalitis, meningitis and transverse myelitis occur frequently in patients with hepatitis E virus (HEV) infection, and this study was conducted to better characterize the role of HEV in the pathogenesis of neurologic disorders. Genotype 4 strain of swine HEV was used to inoculate Mongolian gerbils. Reverse transcription-nested polymerase chain reaction (RT-nPCR), ELISA, histopathology, ultrastructural pathology and enzyme immunohistochemistry method were conducted to investigate the replication and localization of HEV in the central nervous system (CNS) and the consequent pathological changes. Both positive- and negative-strand HEV RNA was detectable in brain and spinal cord from 7 to 28 dpi (days postinoculation) via RT-nPCR. Various pathological changes such as perineural invasion, neuron necrosis, microglia nodule, lymphocyte infiltration, perivascular cuff and myelin degeneration were observed in HEV-positive brains and spinal cords. Immunohistochemical (IHC) staining targeting on HEV ORF2 protein revealed positive signals concentrated mainly in the cytoplasm of neuron, ependymal epithelium and choroid plexus area. Positive area density of ZO-1 (zonula occludens-1) in brain of HEV-positive gerbils decreased, while the GFAP (glial fibrillary acidic protein) expression was upregulated compared with control groups. These results provide strong evidence that HEV is able to damage the blood-brain barrier (BBB), replicate in brain and spinal cord, and hammer the causative role of HEV in the pathogenesis of neurologic disorders.

Hepatitis E virus (HEV) infection has been associated with a wide range of extrahepatic manifestations, so this study was designed to examine the effect and role of HEV on structural and molecular changes in the testicular tissues of Mongolian gerbils experimentally infected with swine HEV. HEV RNA was first detected in testis at 14 days post-inoculation and reached a peak between 28 and 42 days later with viral load between 3.12 and 6.23 logs/g by PCR assays. Changes including vacuolation, sloughing of germ cells, formation of multinuclear giant cells, degeneration, necrosis of tubules and damaged blood-testis barrier were observed through transmission electron microscopy. HEV ORF2 antigen was detected in the sperm cell cytoplasm along with decrease in relative protein of zonula occludens-1 through immunohistochemistry. HEV ORF3 antigen and ZO-1 protein were detectable by Western blotting. Lower (P<.05) serum testosterone and higher (P<.05) blood urea nitrogen level was observed in inoculated Mongolian gerbils. Likewise, increased (P<.05) germ cell apoptosis rate was detected with significant increased expression of Fas-L and Fas in HEV-inoculated groups at each time points. Up-regulation (P<.05 or P<.01) in mRNA level of Fas-L, Fas, Bax, Bcl-2 and caspase-3 was observed in HEV RNA-positive testes. Our study demonstrated that after experimental inoculation, HEV can be detected in testis tissues and viral proteins produce structural and molecular changes that in turn disrupt the blood-testis barrier and induce germ cell apoptosis.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

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.