Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Emerson, Suzanne U.; Nguyen, Hanh; Torian, Udana; Burke, Danielle; Engle, Ronald E.; Purcell, Robert H.

doi:10.1128/jvi.00593-10

Cited by 151 publications

(153 citation statements)

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“…ORF3 protein is required for virus egress, perhaps through interactions with one or more cellular proteins (13,14). Because the Sar-55 and Kernow-C1 ORF3 proteins differ by 17.5% (20 of 114 amino acids), Kernow-C1, but not Sar-55 ORF3 may be able to interact efficiently with pig cellular proteins potentially involved in virus exit; thus, the replication cycle of Sar-55 would be aborted.…”

Section: Discussionmentioning

confidence: 99%

“…Both genotype 1 (14) and genotype 3 (13) viruses produced in cell culture differ significantly from those excreted in the feces, in that they contain ORF3 protein and their virions are not precipitated by anti-ORF2 antibody that readily precipitates fecal virions. If these differences in virus structure or composition are responsible for the reduced specific infectivity of the cultured viruses, it may be impossible to develop a truly robust cell culture system for HEV.…”

Section: Discussionmentioning

confidence: 99%

“…ORF1 occupies the 5′ two-thirds of the genome and encodes enzymes required for RNA replication. ORF2 encodes the structural protein comprising the virion capsid, and ORF3, which mostly overlaps ORF2, encodes a short protein of 114 amino acids that is required for virus egress from cells and that is proposed to perturb numerous cellular pathways (13)(14)(15). ORF2 and ORF3 proteins are translated from a single, bicistronic, subgenomic mRNA in which the ORF3 methionine initiation codon is located 11 nucleotides upstream of that of ORF2 (16).…”

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confidence: 99%

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Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

Shukla

Nguyen

Torian

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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274

326

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The RNA virus, hepatitis E virus (HEV) is the most or second-most important cause of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. In these regions it is an important cause of acute liver failure, especially in pregnant women who have a mortality rate of 20–30%. Until recently, hepatitis E was rarely identified in industrialized countries, but Hepatitis E now is reported increasingly throughout Western Europe, some Eastern European countries, and Japan. Most of these cases are caused by genotype 3, which is endemic in swine, and these cases are thought to be zoonotically acquired. However, transmission routes are not well understood. HEV that infect humans are divided into nonzoonotic (types 1, 2) and zoonotic (types 3, 4) genotypes. HEV cell culture is inefficient and limited, and thus far HEV has been cultured only in human cell lines. The HEV strain Kernow-C1 (genotype 3) isolated from a chronically infected patient was used to identify human, pig, and deer cell lines permissive for infection. Cross-species infections by genotypes 1 and 3 were studied with this set of cultures. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

Shukla

Nguyen

Torian

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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274

326

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“…ORF2 codes for the viral capsid protein. ORF3 encodes a small multifunctional phosphoprotein that interacts with host cellular proteins and may have a role in HEV virion release from the host cell (8)(9)(10). ORF1 encodes an approximately 186-kDa polyprotein with the following domain structure: NH 2 -methyltransferase (11), cysteine protease (12,13), polyproline hypervariable region (HVR) (14,15), ADP-ribose phosphorylase (X or macro domain), helicase (16,17), and RNA-dependent RNA polymerase (RdRp) (18)-COOH.…”

mentioning

confidence: 99%

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

Kenney

Meng

2015

J Virol

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Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCEHEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future. Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in many parts of the world (1, 2). Major outbreaks and epidemics are most common in developing countries, where a lack of proper sanitation conditions leads to waterborne spread of the disease. In industrialized countries, HEV infection is typically sporadic and endemic. Recently, chronic HEV infection has become an important clinical problem in immunosuppressed individual...

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“…We have previously infected several cell lines of human origin such as HepG2, PLC/PRF/5, Huh7, S10-3, Caco2 and A549 with genotype 1 virus purified from human stool samples to see their permissiveness (Devhare et al, 2013) and noticed that HepG2/C3A cells become positive for negative strand 8 h post-infection and IFA positive on the 6th day. HepG2/C3A cells have been successfully used by other researchers for HEV infectivity studies (Emerson et al, 2010;Feagins et al, 2011). Virus particles generated from the chimera HEV-1 (HEV-4 ORF1) would have capsid protein of genotype 1.…”

Section: ¢ and 3¢ Ncrs Dictate Replication Efficiencymentioning

confidence: 99%

Hepatitis E virus (HEV)-1 harbouring HEV-4 non-structural protein (ORF1) replicates in transfected porcine kidney cells

Chatterjee

Devhare

Pingle

et al. 2016

Journal of General Virology

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Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a major public health problem in India. There are four mammalian HEV genotypes worldwide. In India, genotype 1 (HEV-1) is restricted to humans whereas genotype 4 (HEV-4) circulates in pigs. Studies from our laboratory have shown that HEV-4 (swine) virus can establish experimental infection in rhesus monkeys; however, HEV-1 (human) virus cannot infect pigs. Viral and/or cellular factors responsible for this host specificity are not yet known. We developed 12 different genotype 1-4 chimeric full genome clones with pSK-HEV2 as the backbone and by replacing structural (ORF2 and ORF3), nonstructural (ORF1) and non-coding regions (NCR) with corresponding segments from the HEV-4 clone. S10-3 (human hepatoma) and PK-15 (pig kidney) cells were transfected with transcripts generated from the above clones to test their replication competence. Transfected cells were monitored for successful virus replication by detecting replicative intermediate RNA and capsid protein (immunofluorescence assay). All the chimeric constructs were able to replicate in S10-3 cells. However, only two chimeric clones, HEV-1 (HEV-4 5¢NCR-ORF1) and HEV-1 (HEV-4 ORF1), containing 5¢NCR-ORF1 and ORF1 regions from the HEV-4 clone, respectively, were able to replicate in PK-15 cells. We demonstrate for the first time the crucial role of ORF1 polyprotein in crossing the species barrier at the cellular level. These results indicate the importance of interactions between ORF1 protein domains and host cell specific factors during HEV replication and the critical role of cellular factors as post-entry barrier/s in virus establishment.

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Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Cited by 151 publications

References 28 publications

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

Hepatitis E virus (HEV)-1 harbouring HEV-4 non-structural protein (ORF1) replicates in transfected porcine kidney cells

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