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Lack of processing of the expressed ORF1 gene product of hepatitis E virus.

Suppiah S, Zhou Y, Frey TK - Virol. J. (2011)

Bottom Line: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed.However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved.The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA, USA.

ABSTRACT

Background: Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group 1 provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system.

Findings: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease 2 had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved.

Conclusions: The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.

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Related in: MedlinePlus

Schematic diagram of expressed HEV ORF1 and multiple sequence alignment of the putative PCP domain in genotypes 1-4. ORF1 of HEV is shown schematically as a box containing a number of motifs identified by computer-assisted homology searching [2]. The motifs are: methyl/guanylyl transferase (M), Y domain (unknown function), papain-like cysteine protease (PCP?, the presence of which is tested in this study), proline rich region (P), X domain (poly ADP ribose phosphatase), helicase (H) and RNA-dependent RNA polymerase domain (R). For expression, ORF1 was amplified from pTM1HEV, a plasmid used to express ORF1 of the Burma strain of HEV (genotype 1) in our earlier study [1], and pSHEV3, an infectious cDNA clone of the swine US strain of HEV (genotype 3)[14](obtained from X.J. Meng), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF. The multiple sequence alignment consists of the putative PCP of representative members of the four HEV genotypes (Genotype 1, Burma strain M73218; Genotype 2, Mexico strain M74506; Genotype 3, swine US strain AF082843; Genotype 4, China T1 strain AJ272108). An alignment of 135 HEV ORF1 from HEV genomic sequences available on GenBank revealed that the putative cysteine catalytic residue (C483, boxed) is conserved while the putative catalytic histidine residue (H590, boxed) is present in genotype 1 sequences, but is not conserved in the other genotypes. It should be noted that the catalytic cysteine and histidine residues of the PCP in the NS-ORF of rubella virus are conserved in all eight rubella virus genotypes (Yumei Zhou, unpublished data).
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Figure 1: Schematic diagram of expressed HEV ORF1 and multiple sequence alignment of the putative PCP domain in genotypes 1-4. ORF1 of HEV is shown schematically as a box containing a number of motifs identified by computer-assisted homology searching [2]. The motifs are: methyl/guanylyl transferase (M), Y domain (unknown function), papain-like cysteine protease (PCP?, the presence of which is tested in this study), proline rich region (P), X domain (poly ADP ribose phosphatase), helicase (H) and RNA-dependent RNA polymerase domain (R). For expression, ORF1 was amplified from pTM1HEV, a plasmid used to express ORF1 of the Burma strain of HEV (genotype 1) in our earlier study [1], and pSHEV3, an infectious cDNA clone of the swine US strain of HEV (genotype 3)[14](obtained from X.J. Meng), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF. The multiple sequence alignment consists of the putative PCP of representative members of the four HEV genotypes (Genotype 1, Burma strain M73218; Genotype 2, Mexico strain M74506; Genotype 3, swine US strain AF082843; Genotype 4, China T1 strain AJ272108). An alignment of 135 HEV ORF1 from HEV genomic sequences available on GenBank revealed that the putative cysteine catalytic residue (C483, boxed) is conserved while the putative catalytic histidine residue (H590, boxed) is present in genotype 1 sequences, but is not conserved in the other genotypes. It should be noted that the catalytic cysteine and histidine residues of the PCP in the NS-ORF of rubella virus are conserved in all eight rubella virus genotypes (Yumei Zhou, unpublished data).

Mentions: Hepatitis E virus (HEV) is the sole member of the genus Hepevirus belonging to the family Hepeviridae [reviewed in [3]]. HEV is a non-enveloped, plus-sense, single-stranded RNA virus whose genome is approximately 7.2 kb in length and consists of three open reading frames (ORFs): the 5' proximal ORF1 of ~5100 nt which encodes the viral replicase components; the 3' proximal ORF2 of ~2000 nt which encodes the capsid protein; and ORF3 of ~400 nt, which encodes a phosphoprotein of unknown function. ORF2 and ORF3 overlap and are translated from a single subgenomic RNA species. Computer-assisted sequence alignment of the deduced translation product of ORF1 with replicase proteins of other animal and plant plus-strand RNA viruses led to the identification of several common motifs, including putative methyl/guanylyl-transferase, papain-like cysteine protease (PCP), poly ADP ribose phosphatase, helicase and RNA-dependent-RNA-polymerase (RDRP) domains [2](see Figure 1). Of these, activity of the methyl/guanylyl transferase, the helicase, and the RDRP domains have been experimentally demonstrated [4-7].


Lack of processing of the expressed ORF1 gene product of hepatitis E virus.

Suppiah S, Zhou Y, Frey TK - Virol. J. (2011)

Schematic diagram of expressed HEV ORF1 and multiple sequence alignment of the putative PCP domain in genotypes 1-4. ORF1 of HEV is shown schematically as a box containing a number of motifs identified by computer-assisted homology searching [2]. The motifs are: methyl/guanylyl transferase (M), Y domain (unknown function), papain-like cysteine protease (PCP?, the presence of which is tested in this study), proline rich region (P), X domain (poly ADP ribose phosphatase), helicase (H) and RNA-dependent RNA polymerase domain (R). For expression, ORF1 was amplified from pTM1HEV, a plasmid used to express ORF1 of the Burma strain of HEV (genotype 1) in our earlier study [1], and pSHEV3, an infectious cDNA clone of the swine US strain of HEV (genotype 3)[14](obtained from X.J. Meng), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF. The multiple sequence alignment consists of the putative PCP of representative members of the four HEV genotypes (Genotype 1, Burma strain M73218; Genotype 2, Mexico strain M74506; Genotype 3, swine US strain AF082843; Genotype 4, China T1 strain AJ272108). An alignment of 135 HEV ORF1 from HEV genomic sequences available on GenBank revealed that the putative cysteine catalytic residue (C483, boxed) is conserved while the putative catalytic histidine residue (H590, boxed) is present in genotype 1 sequences, but is not conserved in the other genotypes. It should be noted that the catalytic cysteine and histidine residues of the PCP in the NS-ORF of rubella virus are conserved in all eight rubella virus genotypes (Yumei Zhou, unpublished data).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Schematic diagram of expressed HEV ORF1 and multiple sequence alignment of the putative PCP domain in genotypes 1-4. ORF1 of HEV is shown schematically as a box containing a number of motifs identified by computer-assisted homology searching [2]. The motifs are: methyl/guanylyl transferase (M), Y domain (unknown function), papain-like cysteine protease (PCP?, the presence of which is tested in this study), proline rich region (P), X domain (poly ADP ribose phosphatase), helicase (H) and RNA-dependent RNA polymerase domain (R). For expression, ORF1 was amplified from pTM1HEV, a plasmid used to express ORF1 of the Burma strain of HEV (genotype 1) in our earlier study [1], and pSHEV3, an infectious cDNA clone of the swine US strain of HEV (genotype 3)[14](obtained from X.J. Meng), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF. The multiple sequence alignment consists of the putative PCP of representative members of the four HEV genotypes (Genotype 1, Burma strain M73218; Genotype 2, Mexico strain M74506; Genotype 3, swine US strain AF082843; Genotype 4, China T1 strain AJ272108). An alignment of 135 HEV ORF1 from HEV genomic sequences available on GenBank revealed that the putative cysteine catalytic residue (C483, boxed) is conserved while the putative catalytic histidine residue (H590, boxed) is present in genotype 1 sequences, but is not conserved in the other genotypes. It should be noted that the catalytic cysteine and histidine residues of the PCP in the NS-ORF of rubella virus are conserved in all eight rubella virus genotypes (Yumei Zhou, unpublished data).
Mentions: Hepatitis E virus (HEV) is the sole member of the genus Hepevirus belonging to the family Hepeviridae [reviewed in [3]]. HEV is a non-enveloped, plus-sense, single-stranded RNA virus whose genome is approximately 7.2 kb in length and consists of three open reading frames (ORFs): the 5' proximal ORF1 of ~5100 nt which encodes the viral replicase components; the 3' proximal ORF2 of ~2000 nt which encodes the capsid protein; and ORF3 of ~400 nt, which encodes a phosphoprotein of unknown function. ORF2 and ORF3 overlap and are translated from a single subgenomic RNA species. Computer-assisted sequence alignment of the deduced translation product of ORF1 with replicase proteins of other animal and plant plus-strand RNA viruses led to the identification of several common motifs, including putative methyl/guanylyl-transferase, papain-like cysteine protease (PCP), poly ADP ribose phosphatase, helicase and RNA-dependent-RNA-polymerase (RDRP) domains [2](see Figure 1). Of these, activity of the methyl/guanylyl transferase, the helicase, and the RDRP domains have been experimentally demonstrated [4-7].

Bottom Line: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed.However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved.The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Georgia State University, Atlanta, GA, USA.

ABSTRACT

Background: Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group 1 provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system.

Findings: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease 2 had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved.

Conclusions: The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.

Show MeSH
Related in: MedlinePlus