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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.

Show MeSH

Related in: MedlinePlus

Expression of US swine HEV ORF1 and rubella virus non-structural ORF. 293T cells were transfected with 5 μg of pCMV-SHEV DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 12, 24, and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. The cells were washed twice with PBS and lysed with 1X NP-40 buffer (1% NP-40, 150 mM Nacl, 50 mM Tris-HCl at pH 7.4 and 2 mM EDTA) in the presence of 1X complete mini, EDTA-free protease inhibitors (Roche). The lysates were resolved on 8% SDS-PAGE gels followed by transfer of the contents to nitrocellulose membranes and immunoblotted with for 1 h with anti-FLAG antibody-peroxidase conjugate from Sigma (Panel A) or anti-HA antibody- peroxidase conjugate from Roche (Panel B). Membranes were washed 5 times with 0.05% T-TBS (0.5 ml Tween-20 in 1 L 1X TBS [20 mM Tris-HCl at pH 7.5 and 175 mM NaCl]). The peroxidase was detected using BM Blue POD substrate (Roche). Lane1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane2: Mock transfected cells; Lanes 3-5: pCMV-SHEV-transfected cells harvested at 12, 24, and 48 hrs post-transfection (the 185 kDa ORF1 translation product is denoted on the right margin). As a control, the nonstructural protein ORF (NS-ORF) of rubella virus was amplified from Robo502, an infectious cDNA clone (18), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF and cloned into VR1012 plasmid vector; the resulting construct was termed pCMV-NS-ORF. 293T cells were transfected with 5 μg of pCMV-NS-ORF DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 24 and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. Western blotting and probing of lysates was done as described in the legend to Figure 2. Lane 1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane 2: Mock transfected cells; Lanes 3-4: pCMV-NS-ORF-transfected cells harvested at 24 and 48 hrs post-transfection. The 240 kDa precursor and the P150 (N-terminal) and P90 (C-terminal) products are denoted in the right margin.
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Figure 2: Expression of US swine HEV ORF1 and rubella virus non-structural ORF. 293T cells were transfected with 5 μg of pCMV-SHEV DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 12, 24, and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. The cells were washed twice with PBS and lysed with 1X NP-40 buffer (1% NP-40, 150 mM Nacl, 50 mM Tris-HCl at pH 7.4 and 2 mM EDTA) in the presence of 1X complete mini, EDTA-free protease inhibitors (Roche). The lysates were resolved on 8% SDS-PAGE gels followed by transfer of the contents to nitrocellulose membranes and immunoblotted with for 1 h with anti-FLAG antibody-peroxidase conjugate from Sigma (Panel A) or anti-HA antibody- peroxidase conjugate from Roche (Panel B). Membranes were washed 5 times with 0.05% T-TBS (0.5 ml Tween-20 in 1 L 1X TBS [20 mM Tris-HCl at pH 7.5 and 175 mM NaCl]). The peroxidase was detected using BM Blue POD substrate (Roche). Lane1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane2: Mock transfected cells; Lanes 3-5: pCMV-SHEV-transfected cells harvested at 12, 24, and 48 hrs post-transfection (the 185 kDa ORF1 translation product is denoted on the right margin). As a control, the nonstructural protein ORF (NS-ORF) of rubella virus was amplified from Robo502, an infectious cDNA clone (18), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF and cloned into VR1012 plasmid vector; the resulting construct was termed pCMV-NS-ORF. 293T cells were transfected with 5 μg of pCMV-NS-ORF DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 24 and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. Western blotting and probing of lysates was done as described in the legend to Figure 2. Lane 1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane 2: Mock transfected cells; Lanes 3-4: pCMV-NS-ORF-transfected cells harvested at 24 and 48 hrs post-transfection. The 240 kDa precursor and the P150 (N-terminal) and P90 (C-terminal) products are denoted in the right margin.

Mentions: It was not clear why putative processing of ORF1 of the Burma strain differed between the two expression systems. However an infectious cDNA clone was never assembled from the Burma strain of HEV and therefore to guard against artifacts due to potential mutations in the ORF1 construct assembled from this strain, we expressed ORF1 from pSHEV3, a cDNA clone of the US swine strain (genotype 3) that was shown to be infectious [14]. ORF1 from pSHEV3 was introduced into the plasmid vector with N-terminal FLAG and C-terminal HA epitope tags, resulting in a construct termed pCMV-SHEV. As shown in Figure 2A and 2B, expression of the 185 kDa product was detected with both anti-FLAG and anti-HA antibody but no other ORF1-specific products were apparent (background bands of ~95, 90, and 55 kDa were detected by the anti-HA antibody).


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

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

Expression of US swine HEV ORF1 and rubella virus non-structural ORF. 293T cells were transfected with 5 μg of pCMV-SHEV DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 12, 24, and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. The cells were washed twice with PBS and lysed with 1X NP-40 buffer (1% NP-40, 150 mM Nacl, 50 mM Tris-HCl at pH 7.4 and 2 mM EDTA) in the presence of 1X complete mini, EDTA-free protease inhibitors (Roche). The lysates were resolved on 8% SDS-PAGE gels followed by transfer of the contents to nitrocellulose membranes and immunoblotted with for 1 h with anti-FLAG antibody-peroxidase conjugate from Sigma (Panel A) or anti-HA antibody- peroxidase conjugate from Roche (Panel B). Membranes were washed 5 times with 0.05% T-TBS (0.5 ml Tween-20 in 1 L 1X TBS [20 mM Tris-HCl at pH 7.5 and 175 mM NaCl]). The peroxidase was detected using BM Blue POD substrate (Roche). Lane1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane2: Mock transfected cells; Lanes 3-5: pCMV-SHEV-transfected cells harvested at 12, 24, and 48 hrs post-transfection (the 185 kDa ORF1 translation product is denoted on the right margin). As a control, the nonstructural protein ORF (NS-ORF) of rubella virus was amplified from Robo502, an infectious cDNA clone (18), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF and cloned into VR1012 plasmid vector; the resulting construct was termed pCMV-NS-ORF. 293T cells were transfected with 5 μg of pCMV-NS-ORF DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 24 and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. Western blotting and probing of lysates was done as described in the legend to Figure 2. Lane 1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane 2: Mock transfected cells; Lanes 3-4: pCMV-NS-ORF-transfected cells harvested at 24 and 48 hrs post-transfection. The 240 kDa precursor and the P150 (N-terminal) and P90 (C-terminal) products are denoted in the right margin.
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Figure 2: Expression of US swine HEV ORF1 and rubella virus non-structural ORF. 293T cells were transfected with 5 μg of pCMV-SHEV DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 12, 24, and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. The cells were washed twice with PBS and lysed with 1X NP-40 buffer (1% NP-40, 150 mM Nacl, 50 mM Tris-HCl at pH 7.4 and 2 mM EDTA) in the presence of 1X complete mini, EDTA-free protease inhibitors (Roche). The lysates were resolved on 8% SDS-PAGE gels followed by transfer of the contents to nitrocellulose membranes and immunoblotted with for 1 h with anti-FLAG antibody-peroxidase conjugate from Sigma (Panel A) or anti-HA antibody- peroxidase conjugate from Roche (Panel B). Membranes were washed 5 times with 0.05% T-TBS (0.5 ml Tween-20 in 1 L 1X TBS [20 mM Tris-HCl at pH 7.5 and 175 mM NaCl]). The peroxidase was detected using BM Blue POD substrate (Roche). Lane1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane2: Mock transfected cells; Lanes 3-5: pCMV-SHEV-transfected cells harvested at 12, 24, and 48 hrs post-transfection (the 185 kDa ORF1 translation product is denoted on the right margin). As a control, the nonstructural protein ORF (NS-ORF) of rubella virus was amplified from Robo502, an infectious cDNA clone (18), by PCR using primers that added a FLAG epitope at the N-terminus and an HA-epitope at the C-terminus of the ORF and cloned into VR1012 plasmid vector; the resulting construct was termed pCMV-NS-ORF. 293T cells were transfected with 5 μg of pCMV-NS-ORF DNA using Lipofectamine-2000 (as recommended by manufacturer's protocol) and harvested 24 and 48 hours post-transfection. Mock transfected cells were processed similarly as a negative control. Western blotting and probing of lysates was done as described in the legend to Figure 2. Lane 1: Broad range molecular weight standard marker (Mr's in kDa given on left margin); Lane 2: Mock transfected cells; Lanes 3-4: pCMV-NS-ORF-transfected cells harvested at 24 and 48 hrs post-transfection. The 240 kDa precursor and the P150 (N-terminal) and P90 (C-terminal) products are denoted in the right margin.
Mentions: It was not clear why putative processing of ORF1 of the Burma strain differed between the two expression systems. However an infectious cDNA clone was never assembled from the Burma strain of HEV and therefore to guard against artifacts due to potential mutations in the ORF1 construct assembled from this strain, we expressed ORF1 from pSHEV3, a cDNA clone of the US swine strain (genotype 3) that was shown to be infectious [14]. ORF1 from pSHEV3 was introduced into the plasmid vector with N-terminal FLAG and C-terminal HA epitope tags, resulting in a construct termed pCMV-SHEV. As shown in Figure 2A and 2B, expression of the 185 kDa product was detected with both anti-FLAG and anti-HA antibody but no other ORF1-specific products were apparent (background bands of ~95, 90, and 55 kDa were detected by the anti-HA antibody).

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