Three Amino Acid Mutations (F51L, T59A, and S390L) in the Capsid Protein of the Hepatitis E Virus Collectively Contribute to Virus Attenuation

Abstract

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, and the mechanisms of HEV replication and pathogenesis are largely unknown. We previously identified an attenuated genotype 3 HEV mutant (pSHEV-1) containing three unique amino acid mutations (F51L, T59A, and S390L) in the capsid protein. To determine the role of each of these mutations, we constructed three HEV single mutants (rF51L, rT59A, and rS390L) which were all found to be replication competent in Huh7 liver cells. To determine the pathogenicities of the mutants, we utilized the specific-pathogen-free (SPF) pig model for HEV and a unique inoculation procedure that bypasses the need for propagating infectious HEV in vitro. A total of 60 pigs were intrahepatically inoculated, via an ultrasound-guided technique, with in vitro-transcribed full-length capped RNA transcripts from the infectious clones of each single mutant, the pSHEV-1 triple mutant, wild-type pSHEV-3, or phosphate-buffered saline (PBS) buffer (n ‫؍‬ 10). The results showed that the F51L mutation partially contributed to virus attenuation, whereas the T59A and S390L mutations resulted in more drastic attenuation of HEV in pigs, as evidenced by a significantly lower incidence of viremia, a delayed appearance and shorter duration of fecal virus shedding and viremia, and lower viral loads in liver, bile, and intestinal content collected at three different necropsy times. The results indicate that the three mutations in the capsid protein collectively contribute to HEV attenuation. This study has important implications for developing a modified live-attenuated vaccine against HEV.Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis in many developing countries in Asia, the Middle East, and Africa and in Mexico (2,13,[40][41][42]49), although the disease has also been reported in many industrialized countries, including the United States (53). HEV is transmitted primarily by the fecal-oral route through contaminated drinking water or food. The disease generally affects young adults and has a mortality rate of less than 1% in the general population, although a significantly higher rate of mortality, up to 30%, has been reported in infected pregnant women (47). HEV is a small nonenveloped virus, and its genome is a single-strand, positive-sense RNA molecule of approximately 7.2 kb in size. Currently, HEV is classified in the genus Hepevirus of the family Hepeviridae (9).The genomic RNA of HEV contains a short 5Ј noncoding region (NCR), three open reading frames (ORF1, -2, and -3), and a 3Ј NCR (22). A cap structure has been identified at the 5Ј end of the viral genome and is required for efficient virus replication in vivo (14). The ORF2 and ORF3 proteins are translated from a bicistronic subgenomic mRNA (18,25). ORF1 encodes a nonstructural protein with multiple functional domains, including methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase (RdRp) (29). It remains unclear whether the ORF1 polyprotein func...

Hepatitis E Virus Replication Requires an Active Ubiquitin-Proteasome System

“…Briefly, Huh7 cells were transfected with the full-length capped RNA transcripts of the pSK-HEV2 infectious clone, and capsid protein synthesis was monitored by IFA (8). When 1 M MG132 inhibitor was added to culture medium at 24 h posttransfection, no capsid protein synthesis was detected by IFA ( Fig.…”

Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication

“…At 5 days posttransfection, LMH cells were trypsinized and replated on 24-well plates. On day 6, the LMH cells were rinsed with phosphate-buffered saline (PBS), fixed with a solution containing 70% acetone and 30% ethanol, and stained by immunofluorescence assay (IFA) with a 1:500-diluted anti-avian HEV convalescent-phase serum as previously described (8,15,32). For Huh7 cells, at 3 days posttransfection, the cells were trypsinized and replated onto wells of LabTek chamber slides.…”

“…LMH chicken liver cells, which support the replication of avian HEV (15), were transfected at approximately 85% confluence with RNA transcripts generated from avian HEV replicon constructs in a 12-well plate by using a Lipofectamine LTX kit (Invitrogen) essentially as previously described (15,32). The replication competency of HEV chimeras with swapped HVRs was determined by transfecting 10 g of the transcribed RNA from each of the chimeras as well as wild-type HEV into Huh7 cells, using the DMRIE-C transfection reagent as previously described (8).…”

“…For Huh7 cells, at 3 days posttransfection, the cells were trypsinized and replated onto wells of LabTek chamber slides. On day 6, the Huh7 cells were rinsed with PBS, fixed with acetone, and stained by IFA with a 1:200-diluted chimpanzee anti-HEV convalescent-phase serum (chimp 1313 serum) as previously described (8,9). Vectashield (Vector Laboratories) mounting medium was added to the washed wells and viewed under a Zeiss LSM 510 laser scanning confocal microscope.…”

“…The in vitro-generated capped RNA transcripts were used for transfection of Huh7-S10-3 cells and LMH cells as previously described (8,15,32). For transfection of Huh7 cells with RNA transcripts generated from plasmid constructs with the backbone of the genotype 1 human HEV replicon (firefly luciferase replicon), the RNA transcripts synthesized from the T7 promoter of a Renilla luciferase vector (pRL-TK; Promega) were used for normalization and as an internal control.…”