Synthesis, Solid State NMR and Powder X-Ray Diffraction Studies on Ethane-1,2-Dithiolatobismuth(III) Alkyl Dithiocarbonates

<div><p>GRAPHICAL ABSTRACT</p><p></p></div

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus

Deletion mapping of interaction domains in ORF4 for binding with g-1 ORF3, X and Helicase.

<p>Deletion mapping of interaction domains in ORF4 for binding with g-1 ORF3, X and Helicase.</p

Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.

<p>Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.</p

An IRESl (Internal ribosome entry site-like) element located upstream of ORF4 coding sequence drives its translation independent of ORF1.

<p><b>(A)</b> Predicted secondary structure of IRESl using “mfold”. Cyan letters indicate core IRESl sequence. A, B, C: stem loops, A*: bulge. Substitutions that impair IRESl are in bold. <b>(B)</b> Organization of Dual luciferase reporter plasmid. <b>(C)</b> Dual luciferase reporter assay. Values are mean±SEM.</p

ORF4 antibody is detected in HEV patients and its over expression enhances viral replication.

<p><b>(A)</b> Top: Purified GST-ORF4 stained with coomassie blue (left most) and western of equal aliquots of the same using healthy (CS1, CS2) and acute HEV infected (KU168733-KU168737) patient sera. Bottom: Top blots reprobed with anti-ORF4 antibody. <b>(B)</b> Western of whole cell extract from indicated cells using ORF4 and GAPDH antibodies. <b>(C)</b> QRT-PCR of sense and anti-sense RNA in pCDNA5-Huh7 and ORF4-Huh7 cells transfected with <i>in vitro</i> synthesized wild type (WT) or mutant HEV genome and treated as indicated.<b>(D)</b> Quantitation of viral ORF1 (helicase) and ORF2 expression in pCDNA5-Huh7 and ORF4-Huh7 cells, transfected with wildtype (WT HEV) or ORF4 expression deficient mutant (DM HEV) HEV genomic RNA and treated with the indicated compounds. Ten random fields of approximately 30 cells in each field were counted for helicase, ORF2, DAPI (nuclear stain) fluorescence and percentage ±SEM calculated.</p

G-3 RdRp, X and Helicase associate with each other in Huh7 cells.

<p><b>(A)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp and X, immunoprecipitated and revealed using indicated antibodies. <b>(B)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 Helicase and X, immunoprecipitated and revealed using indicated antibodies. <b>(C)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp and Helicase, immunoprecipitated and revealed using indicated antibodies. <b>(D)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp, Helicase and X, immunoprecipitated and revealed using indicated antibodies.</p

ORF4 is a target of host ubiquitin-proteasome machinery.

<p><b>(A)</b> Western blot using anti-Flag (top) and anti-GAPDH (bottom). <b>(B)</b> Western blot using anti-ubiquitin (top) and anti-Flag (bottom). <b>(C)</b> Anti-Flag western blot of Huh7 cells expressing WT and K51N mut ORF4, treated with cycloheximide (panel 1, 3). Same blots were reprobed with anti-GAPDH (panel 2, 4). <b>(D)</b> Immunofluorescence of ORF4 in Huh7 cells transfected with <i>in vitro</i> synthesized WT HEV or K51N HEV. Scale: 20μm. Shown are merged images of nuclei (blue) and ORF4 (green). “→”: positive staining, “►”: unstained. <b>(E)</b> QRT-PCR of HEV sense and anti-sense RNA from Huh7 cells transfected and treated as indicated. Veh: Vehicle, TUN:tunicamycin.</p