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Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1β expression in the serum and liver. Whereas, the viral infection in IL-1β receptor-I deficient (IL-1R1-/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45+Gr-1high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1β secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1β induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words).