Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.


Introduction
Hepatitis E virus (HEV) is a quasi-enveloped (blood or tissue culture) or non-enveloped (feces) virus [1,2]. HEV contains a positive-sense single-strand RNA as the genome and has been classified in the family Hepeviridae [3]. Novel HEV strains have been identified in many animal species in recent years, and the genetic diversity within HEVs has been disclosed [4][5][6][7][8][9]. The current taxonomy shows that the family Hepeviridae includes two subfamilies: Orthohepevirinae and Parahepevirinae. The subfamily Orthohepevirinae includes at least four genera: Paslahepevirus, Rocahepevirus, Chirohepevirus, and Avihepevirus self-limited hepatitis, immunocompromised and transplant patients are vulnerable to prolonged infections and tend to develop chronic hepatitis [14].
The virological information obtained to date concerning ORF4 of HEV-1 is extremely limited, and a study published in 2016 indicated that the ORF4 product of HEV-1 interacts with multiple viral proteins to form a viral replication complex containing RdRp, Hel, and X proteins, and the ORF4-related protein promotes RdRp activity by interacting with host eEF1α1 and tubulin β, which is indispensable for the replication of HEV-1 [19]. However, there has been no report to confirm the role of ORF4 during the natural course of HEV-1 replication and infection. In the present study, we produced ORF4-deficient HEV-1s by a reverse genetic system to investigate whether ORF4 affects the replication and infectivity of HEV-1.  The virological information obtained to date concerning ORF4 of HEV-1 is extremely limited, and a study published in 2016 indicated that the ORF4 product of HEV-1 interacts with multiple viral proteins to form a viral replication complex containing RdRp, Hel, and X proteins, and the ORF4-related protein promotes RdRp activity by interacting with host eEF1α1 and tubulin β, which is indispensable for the replication of HEV-1 [19]. However, there has been no report to confirm the role of ORF4 during the natural course of HEV-1 replication and infection. In the present study, we produced ORF4-deficient HEV-1s by a Viruses 2023, 15, 784 3 of 13 reverse genetic system to investigate whether ORF4 affects the replication and infectivity of HEV-1.

Materials and Methods
2.1. Design of ORF4-Defective HEV-1 and the In Vitro Transcription of HEV-1 RNA An entire genome cDNA of HEV-1 was synthesized based on the nucleotide sequence derived from a subtype 1a strain (GenBank accession no. LC061267). The putative ORF4 of HEV-1 (nucleotides [nt] 2836-3255) encodes 139 amino acids (aa). The start ATG codon was changed to TTG (A2836T) or ACG (T2837C). Because we observed a downstream in-frame ATG codon (nt 2884-2886) near the N-terminal of ORF4, we also designed a mutation, T2885C. The mutations T2837C and T2885C did not result in the ORF1 aa changes, but A2836T resulted in an aa mutation, D937V (Asp to Val) in ORF1 and T2885C resulted in a aa mutation (Met to Thr) in ORF4 ( Figure 1, Table 1). The complete RNA transcripts of the wild-type HEV-1 and four mutant viruses were synthesized, and we added a T7 RNA polymerase promoter sequence to them at the 5 end and an XbaI-site at the 3 end. These five constructs were cloned into pUC57 vector to generate plasmids pUC57-HEV-1, pUC57-A2836T, pUC57-T2837C, pUC57-T2885C, and pUC57-T2837C/T2885C, respectively (GeneScript, Piscataway, NJ, USA) ( Table 1). All plasmids were linearized with XbaI and purified by phenol/chloroform extraction. Capped HEV RNA was synthesized using an mMESSAGE mMACHINE T7 transcription kit (Ambion, Austin, TX, USA) according to the manufacturer's recommendations.
The synthesized capped RNAs were treated with TURBO DNase and purified by lithium chloride precipitation and named HEV-1 RNA , A2836T RNA , T2837C RNA , T2885C RNA , and T2837C/T2885C RNA , respectively (Table 1). HEV-1 RNA encoded the wild-type HEV-1 genome sequence. A2836T RNA lost the ORF4 and gained an aa mutation of D937V in the ORF1. T2837C RNA lost the ORF4 without ORF1 aa mutation. T2885C RNA kept the ORF4 with an aa mutation (M17T) in the ORF4. T2837C/T2885C RNA lost the ORF4 and obtained an aa mutation (M17T) in the ORF4. Capped RNA sequences were confirmed by a next-generation sequencing analysis before transfection.

Cell Culture and Transfection
A human hepatocarcinoma cell line, PLC/PRF/5 (JCRB0406), was grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Nichirei Biosciences, Tokyo), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Grand Island, NY, USA) at 37 • C in a humidified 5% CO 2 atmosphere and passaged every 3 days. For transfection, 5 × 10 5 PLC/PRF/5 cells were cultured in a 25 cm 2 tissue culture flask for 24 h and then washed with phosphate-buffered saline (PBS) and supplemented with 6.6 mL of the new medium with 10% FBS. The transfection was performed using a TransIT-mRNA Transfection Kit (Mirus Bio, Madison, WI, USA). Briefly, 6.6 µg of the capped parental or mutated virus RNAs were combined with 630 µL of Opti-MEM (Gibco), and then 13.2 µL of mRNA Boost Reagent and 13.2 µL of TransIT-mRNA reagent were added to the mixture. After 5 min of incubation at room temperature, the mixture was added to the PLC/PRF/5 cells containing 6.6 mL of medium. After a 12 h incubation at 37 • C, the cells were washed three times with PBS and the medium was replaced with 10 mL of maintenance medium: medium 199 (Invitrogen, Carlsbad, CA, USA) containing 2% (v/v) heat-inactivated FBS and 10 mM MgCl 2 . Further incubation was performed at 36 • C. The medium was replaced with the new medium every 4 days, and the culture supernatant was used for the detection of HEV-1 RNA.

Infection of Mongolian Gerbils and the Sample Collection
Fifteen 6-week-old female Mongolian gerbils (MON/Jms/GbsSlc, SLC, Hamamatsu, Japan) were used. All were individually housed in a Biosafety Level-2 facility and were tested and confirmed to be negative for both the serum anti-HEV IgG antibodies and the HEV RNA. The gerbils were randomly separated into five groups (n = 3 each). Each gerbil was intraperitoneally inoculated with 1 mL of HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, or T2837C/T2885Cp0 containing 10 8 copies/mL of the viral RNA.
Fecal specimens were collected every 3 or 4 days, and 10% stool suspensions were used for the detection of the viral RNA. Serum samples were collected only at the end of the experiment and were used for the detection of the virus RNA, anti-HEV IgG antibodies, and ALT. At the end of the experiment, the gerbils were euthanized by exsanguination from the heart under anesthesia.
The animal experiments were reviewed and approved by the institutional ethics committee of our institution and were performed according to the Guides for Animal Experiments issued by Japan's National Institute of Infectious Diseases under code 121025 (27 May 2021).

Detection of HEV-1 RNA
The extraction of viral RNA from 200 µL of the cell culture supernatants and from the 10% stool suspensions was carried out by a MagNA Pure 96 System (Roche Applied Science, Mannheim, Germany) with a MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Applied Science). HEV-1 RNA was examined by a one-step real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using TaqMan Fast Virus 1-step Master Mix (Applied Biosystems, Foster City, CA, USA) and a QuantStudio 3 Real-Time PCR System (Applied Biosystems). The RT-qPCR was carried out under the condition of 5 min at 50 • C, 20 s incubation at 95 • C, followed by 40 cycles for 3 s at 95 • C and 30 s at 60 • C with a forward primer, JVHEVF (5 -GGTGGTTTCTGGGGTGAC-3 nt 5346-5363), a reverse primer, JVHEVR (5 -AGGGGTTGGTTGGATGAA-3 nt 5393-5415), and a probe, JVHEVP (5 -FAM-TGATTCTCAGCCCTTCGC-TAMRA-3 nt 5369-5386) [21]. A 10-fold serial dilution of the capped HEV-1 RNA (10 7 to 10 1 copies) was used as the standard for the quantitation of the viral genome copy numbers. Amplification data were collected and analyzed with QuantStudio Design & Analysis software ver. 1.5 (Applied Biosystems).
A semi-nested reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify 507 base pairs (bp) of the ORF1 genome (nt 2794-3300) that covered the entire genome of ORF4. Five microliters of the cDNA was used for the first PCR in 50 µL of the reaction mixture containing an external forward primer, ORF4F1 (5 -TATACCAGGTGCCAATCGGT-3 nt 2695-2714) and a reverse primer, ORF4R3 (5 -ACCACGGATCAACTCGCATA-3 nt 3301-3200). Two microliters of the first PCR product was used for the nested PCR with an inner forward primer, ORF4F2 (5 -ACTTGCTGCCAGA TGGTTTG-3 nt 2774-2793) and ORF4R3. The PCR amplification was performed under the following conditions: inoculation at 94 • C for 60 s, followed by 35 cycles of 30 s at 94 • C, 30 s at 55 • C, and 75 s at 72 • C, and a final extension at 72 • C for 7 min. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and the nucleotide sequencing was carried out with primers ORF4F2 and ORF4R3 using an ABI 3130 Genetic Analyzer Automated Sequencer (Applied Biosystems) and a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer's instructions. The sequence analysis was performed using the Genetyx ver. 11.0.4 software program (Genetyx, Tokyo).

Detection of Anti-HEV IgG Antibodies
Anti-HEV IgG antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HEV-1 as the antigen as described [22]. Briefly

Expression of ORF4-Related Protein
The ORF4 genome containing the BamH I site before the start codon and the Not I site after the stop codon was amplified by PCR with the primers ORF4BamH I (5 -GGATCCATGTT GCACGGACAGCGAAT-3 ) and ORF4Not I (5 -GCGGCCGCCTAAGTCGGGCCTGATGG CG-3 ). The amplified DNA fragments were purified with a Qiagen Gel purification kit (Qiagen, Valencia, CA, USA) and cloned into a TA 2.1 cloning vector (Invitrogen, San Diego, CA, USA), then ligated with an expression-vector pET32a (+) to generate pET32a-ORF4 and used to transform an E. coli strain BL21 (DE3). The E. coli was incubated in Luria-Bertani medium containing 100 µg/mL ampicillin at 37 • C until the absorbance at 600 nm reached 0.6. Then, 0.1 mM isopropyl beta-d-thiogalactopyranoside (IPTG) was added to the culture and incubated for 3 h.
The virus RNA was detected in the HEV-1 RNA -transfected cell culture supernatant on day 4 post-transfection (p.t.), at 3.0 × 10 7 copies/mL. The copy numbers temporarily decreased to 2.0 × 10 7 copies/mL on day 8 p.t. and then increased again and reached 1.8 × 10 8 copies/mL on day 24 p.t.; the copy numbers were then maintained at~3.6 × 10 8 to 7.3 × 10 8 copies/mL until day 56 p.t. Similar replication patterns were observed in the T2837C RNA , T2885C RNA , and T2837C/T2885C RNA -transfected cells.
In contrast, the virus RNA was detected in the A2836T RNA -transfected cell culture supernatant on day 4 p.t. at 3.3 × 10 7 copies/mL, and the copy numbers decreased to 2.2 × 10 6 copies/mL on day 12 p.t. and then gradually increased. Although the viral copy numbers increased to 1.3 × 10 8 copies/mL on day 40 p.t. and were maintained at~2.3 × 10 8 to 3.2 × 10 8 copies/mL until day 56 p.t., the RNA level was lower than those detected in the HEV-1 RNA , T2837C RNA , T2885C RNA , and T2837C/T2885C RNA -transfected cells (Figure 2). These results indicated that the mutation of M1T and M17T of ORF4 did not affect the HEV-1 replication in the PLC/PRF/5 cells. In contrast, the mutation of D937V in the ORF1 reduced unequivocally the replication. We designated the viruses recovered from the supernatants as HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0, respectively.
Capped RNAs encoding an entire genome derived from the wild-type HEV-1 a those derived from the ORF4-defective genomes were prepared by in vitro transcripti as described above. The capped viral RNAs, i.e., HEV-1 RNA , A2836T RNA , T2837C R T2885C RNA , and T2837C/T2885C RNA , were used to transfect PLC/PRF/5 cells. T transfections were performed using triplicate samples.
However, the RNA titers of the A2836Tp0-inoculated cells on days 4, 24, and 40 p.i. were 6.88 × 10 5 , 2.16 × 10 7 , and 2.21 × 10 8 copies/mL, respectively, and these values are clearly lower than those detected in the cells infected with the other four viruses (Figure 3). These results further confirmed that two in-frame ATG mutations in ORF4 did not affect the infectivity in PLC/PRF/5 cells, but an accompanying mutation, D937V, in the X-domain of ORF1 reduced the virus replication in PLC/PRF/5 cells. The viruses recovered from the supernatants of p0 virus-infected cells were designated HEV-1p1, A2836Tp1, T2837Cp1, T2885Cp1, and T2837C/T2885Cp1, respectively.

Nucleotide Sequence Analyses of ORF4-Defective HEV-1s
To confirm whether the mutated sequences were stable during the virus replication, we used the HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 collected on day 60 p.t. and HEV-1p1, A2836Tp1, T2837Cp1, T2885Cp1, and T2837C/T2885Cp1 collected on day 44 p.i. for the amplification of 507 bp that covered the entire ORF4 genome (nt 2794-3300) by RT-PCR. The nucleotide sequences from all 30 samples (three samples each from the 10 supernatants) were identical to each respective nucleotide sequence. These results indicated that no mutations occurred in ORF4 and the mutations of A2836T, T2837C, and T2885C were genetically stable during the virus replication in the PLC/PRF/5 cells.

Infectivity of the ORF4-Defective HEV1s In Vivo
For the investigation of the infectivity of the ORF4-defective viruses in vivo, HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 containing 1.0 × 10 8 copies/mL of the virus RNA were intraperitoneally inoculated into Mongolian gerbils as described in the Materials and Methods. The virus RNA in the fecal specimens was monitored by RT-qPCR. As shown in Figure 4a, the virus RNAs were detected in all of the fecal specimens from the HEV-1p0-, T2837Cp0-, T2885Cp0-, and T2837C/T2885Cp0-inoculated gerbils, although the detectable periods differed. The virus RNAs reached peaks around day 14 p.i. and the titers ranged from 1.4 × 10 4 to 3.2 × 10 4 copies/g in the HEV-1p0-inoculated gerbils, from 1.1 × 10 4 to 8.5 × 10 4 copies/g in the T2837Cp0-inoculated gerbils, from 2.4 × 10 4 to 1.4 × 10 5 copies/g in the T2885Cp0-inoculated gerbils, and from 1.6 × 10 4 to 9.3 × 10 4 copies/g in the HEV-

Nucleotide Sequence Analyses of ORF4-Defective HEV-1s
To confirm whether the mutated sequences were stable during the virus replication, we used the HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 collected on day 60 p.t. and HEV-1p1, A2836Tp1, T2837Cp1, T2885Cp1, and T2837C/T2885Cp1 collected on day 44 p.i. for the amplification of 507 bp that covered the entire ORF4 genome (nt 2794-3300) by RT-PCR. The nucleotide sequences from all 30 samples (three samples each from the 10 supernatants) were identical to each respective nucleotide sequence. These results indicated that no mutations occurred in ORF4 and the mutations of A2836T, T2837C, and T2885C were genetically stable during the virus replication in the PLC/PRF/5 cells.

Infectivity of the ORF4-Defective HEV1s In Vivo
For the investigation of the infectivity of the ORF4-defective viruses in vivo, HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 containing 1.0 × 10 8 copies/mL of the virus RNA were intraperitoneally inoculated into Mongolian gerbils as described in the Materials and Methods. The virus RNA in the fecal specimens was monitored by RT-qPCR. As shown in Figure 4a, the virus RNAs were detected in all of the fecal specimens from the HEV-1p0-, T2837Cp0-, T2885Cp0-, and T2837C/T2885Cp0-inoculated gerbils, although the detectable periods differed. The virus RNAs reached peaks around day 14 p.i. and the titers ranged from 1.4 × 10 4 to 3.2 × 10 4 copies/g in the HEV-1p0-inoculated gerbils, from 1.1 × 10 4 to 8.5 × 10 4 copies/g in the T2837Cp0-inoculated gerbils, from 2.4 × 10 4 to 1.4 × 10 5 copies/g in the T2885Cp0-inoculated gerbils, and from 1.6 × 10 4 to 9.3 × 10 4 copies/g in the HEV-T2837C/T2885Cp0-inoculated gerbils. The virus RNA titers then decreased and became undetectable in all of the HEV-inoculated gerbils on day 28 p.i. In contrast, no virus RNA was detected in the three A2836Tp0-inoculated gerbils (Figure 4a).   All of the gerbils were euthanized on day 28 p.i., and serum samples were collected for the detection of the anti-HEV IgG antibodies and ALT. The anti-HEV IgG antibodies were detected in the serum of all of the animals inoculated with HEV-1p0, T2837Cp0, T2885Cp0, or T2837C/T2885Cp0, and the titers were 1:6400-51,200 in the HEV-1p0-inoculated gerbils, 1:6400-12,800 in the T2837Cp0-inoculated gerbils, 1:12,800-51,200 in the HEV-T2885Cp0inoculated gerbils, and 1:3200-1:12,800 in the T2837C/T2885Cp0-inoculated gerbils. No antibody was detected in any of the three A2836Tp0-inoculated animals (Figure 4b).
The ALT values ranged from 41 IU/L to 86 IU/L in the gerbils on day 28 p.i., and there was no significant difference among the HEV-1p0-, T2836Ap0-, T2837Cp0-, T2885Cp0-, and T2837C/T2885Cp0-inoculated animals ( Table 2). To confirm whether infection occurred actually by ORF4-defective HEV-1, we amplified a portion of the virus RNA genome by RT-PCR using the fecal samples. The virus RNApositive stool suspensions collected from T2837Cp0-, T2885Cp0-, and T2837C/T2885Cp0infected gerbils were concentrated by ultracentrifugation and used for the virus RNA extraction; in addition, 507 bp of ORF1 containing entire genome of ORF4 was amplified by RT-PCR. The nucleotide sequence analyses confirmed that no mutations occurred in ORF4, and T2837Cp0 and T2885Cp0, which contain in-frame ATG mutations but no accompanying aa mutation in ORF1, were genetically stable during the replication in the gerbils, and that the putative ORF4 was not essential for HEV infection in vivo. In contrast, A2836Tp0 containing the D937V change in the X-domain of the ORF1 did not infect the gerbils, demonstrating that this mutation was critical for the HEV-1 infectivity.

Detection of Viral Protein in the Wild-type and the ORF4-defective HEV-1s-Infected PLC/PRF/5 Cells
For the investigation of whether the ORF4-encoded protein appeared in the virusinfected cells, we collected HEV-1p0-, HEV-T2837Cp0-, and HEV-T2837C/T2885Cp0infected PLC/PRF/5 cells on day 48 p.i., and the viral proteins were analyzed by WB with monkey anti-HEV-1 serum collected from an HEV-1 (LC061267)-infected cynomolgus monkey [25]. The antibody titer of anti-capsid protein was as high as 1:3,276,800 by ELISA (Figure 5a).
Two protein bands were detected: one~72 kDa corresponding to the capsid protein and the other~13 kDa corresponding to the ORF3 protein ( Figure 5b). However, no other extra protein band was detected in the wild-type virus-infected cells compared to the two ORF4-defective HEV-1-infected cells, demonstrating no evidence of the putative viral protein(s) derived from ORF4 appeared in HEV-1-infected cells.
To confirm whether the antibody against ORF4 was induced in the HEV-1 infected monkeys, we cloned the ORF4 into the vector pET32a (+) and expressed the ORF4 in E. coli strain BL21 (DE3). Since ORF4 was inserted between the BamH I and Not I in the MCS region of pET32a (+), the expressed fusion protein should have 306 aa (167 aa from the vector and 139 aa from ORF4) and the molecular weight was calculated as~33 kDa. As shown in Figure 5c, an approx. 19 kDa protein (p19) derived from the 167 aa was detected in vector pET32a (+)-transformed cells, whereas an approx. 33 kDa protein (p33) was observed in the pET32a-ORF4-transformed cells (Figure 5b). The WB analyses showed that p33 reacted not only with monkey anti-HEV-1 serum (Figure 5d) but also with monkey anti-HEV-7 serum [26] (Figure 5e), suggesting that sera from the HEV-1-infected monkey had non-specific reactivity against p33. region of pET32a (+), the expressed fusion protein should have 306 aa (167 aa from t vector and 139 aa from ORF4) and the molecular weight was calculated as ~33 kDa. shown in Figure 5c, an approx. 19 kDa protein (p19) derived from the 167 aa was detect in vector pET32a (+)-transformed cells, whereas an approx. 33 kDa protein (p33) w observed in the pET32a-ORF4-transformed cells (Figure 5b). The WB analyses show that p33 reacted not only with monkey anti-HEV-1 serum (Figure 5d) but also w monkey anti-HEV-7 serum [26] (Figure 5e), suggesting that sera from the HEV-1-infect monkey had non-specific reactivity against p33. Figure 5. Detection of viral proteins in HEV-1-infected PLC/PRF/5 cells. A cynomolgus monke serum bearing the anti-HEV-1 IgG antibody titer 1:3,276,800 by ELISA was used for Western b analyses. The minimum endpoints of the antibody titers are blackened (a). HEV-1p0-, T2837Cp and T2837C/T2885Cp0-infected PLC/PRF/5 cells were harvested on day 48 p.i., and the vi proteins were detected by Western blot analysis (b). ORF4 was expressed by an E. coli express system, and the related protein was analyzed by SDS-PAGE (c) and a Western blot analysis w monkey anti-HEV-1 serum (d) and anti-HEV-7 serum (e). M: molecular weight, NC: no-infec

Discussion
Although a putative ORF4 has been observed in the HEV-1 genome, its function has not been clear and the information regarding this ORF is limited. A reverse genetic system, which is a powerful tool to produce infectious HEVs from cloned cDNAs, allowed us to explore the function of ORF4 in the present study. We directly mutated the putative initiator methionine in the ORF4, ATG to TTG (M to L) or ATG to ACG (M to T), in order to disable the translation from the ORF4. The results demonstrated that these mutant viruses retained the ability to replicate in PLC/PRF/5 cells as the wild-type HEV-1 did, although a later-developed mutant virus with an accompanying aa mutation (D937V) in ORF1 exhibited slower virus replication, demonstrating that the ORF4 was not essential for