Increased viperin expression induced by avian infectious bronchitis virus inhibits viral replication by restricting cholesterol synthesis: an in vitro study.

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-ß-cyclodextrin (MßCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.

Endoplasmic reticulum stress and proteasome pathway involvement in human podocyte injury with a truncated COL4A3 mutation.

BACKGROUND: Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury. METHODS: Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. RESULTS: Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group. CONCLUSIONS: We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation.

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent, cross-clade protection against H5N1 influenza virus in mice.

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8(+) T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.