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

Citation: Nair VP, Anang S, Subramani C, Madhvi A, Bakshi K, Srivastava A, et al. (2016) Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus. PLoS Pathog 12(4): e1005521. doi:10.1371/journal.ppat.1005521
Published: April 1, 2016

Abstract
Hepatitis_E_virusHepatitis 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.

Author Summary: Hepatitis E virus (HEV) is one of the most common causes of acute and sporadic viral hepatitis. It is a small positive strand RNA virus, which is transmitted through the feco-oral route. Owing to lack of sanitation and unavailibility of safe drinking water, populations of developing and resource starved countries are prone towards HEV infection. Recent reports also indicate HEV induced acute and chronic hepatitis in organ transplant patients. Another peculiar characteristic of HEV is attributed to its ability to cause high mortality (~30%) in infected pregnant women. Even after 30 years of discovery of the virus, little information exists regarding viral life cycle and replication machinery. HEV is divided into seven genotypes. Genotype-3 and 4 viruses infect humans and a few animals (such as pigs, deer, mongeese) and have been reported from industrialized countries. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus, which is known to infect human and is a major public health concern in south Asian countries, replicates poorly in mammalian cell culture and no other efficient model exists to investigate the viral life cycle. With the goal of developing an efficient laboratory model of genotype-1 HEV, we attempted to identify a permissive cellular condition that would allow efficient viral replication in human hepatoma cells. Here, we report that endoplasmic reticulum stress inducing agents promote genotype-1 HEV replication by initiating cap-independent, internal translation mediated synthesis of a novel viral factor, which we have named ORF4. Further investigations revealed that ORF4 is expressed only in genotype-1 and it acts by interacting with multiple viral and host proteins and cooperates with host eEF1α1 (eukaryotic elongation factor 1 isoform 1) to control the activity of viral RNA dependent RNA polymerase. Moreover, a proteasome resistant ORF4 mutant significantly enhanced viral replication. Thus, our study identifies an optimal condition required for efficient replication of genotype-1 HEV and dissects out the molecular mechanism governing that. Data presented here will be instrumental in developing an efficient model of the virus.

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