Abstract
BackgroundHepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, grows poorly in cell culture and in general does not cause cytopathic effect. Foreign sequences have been inserted into different parts of the HAV genome. However, the packaging size limit of HAV has not been determined. The purpose of the present study is to investigate the maximum size of additional sequences that the HAV genome can tolerate without loosing infectivity.ResultsIn vitro T7 polymerase transcripts of HAV constructs containing a 456-nt fragment coding for a blasticidin (Bsd) resistance gene, a 1,098-nt fragment coding for the same gene fused to GFP (GFP-Bsd), or a 1,032-nt fragment containing a hygromycin (Hyg) resistance gene cloned into the 2A-2B junction of the HAV genome were transfected into fetal Rhesus monkey kidney (FRhK4) cells. After antibiotic selection, cells transfected with the HAV construct containing the resistance gene for Bsd but not the GFP-Bsd or Hyg survived and formed colonies. To determine whether this size limitation was due to the position of the insertion, a 606 bp fragment coding for the Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence was cloned into the 5' nontranslated (NTR) region of HAV. The resulting HAV-IRES retained the EMCV IRES insertion for 1-2 passages. HAV constructs containing both the EMCV IRES at the 5' NTR and the Bsd-resistance gene at the 2A-2B junction could not be rescued in the presence of Bsd but, in the absence of antibiotic, the rescued viruses contained deletions in both inserted sequences.ConclusionHAV constructs containing insertions of approximately 500-600 nt but not 1,000 nt produced viable viruses, which indicated that the HAV particles can successfully package approximately 600 nt of additional sequences and maintain infectivity.
Highlights
Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, grows poorly in cell culture and in general does not cause cytopathic effect
Insertions at the 2A-2B junction To determine the packaging size limit of the HAV genome, we inserted foreign sequences into a 66-nt polylinker engineered between 3C is a cysteine proteinase (3Cpro) cleavage sites at the 2A-2B junction of the HAV cDNA in pHAVvec9 [19] (Figure 1)
A 396-nt fragment containing a Bsd resistance gene coding for a Bsd deaminase, a 1098-nt fragment coding for the same resistance gene fused to the green fluorescent protein (GFP) protein (GFP-Bsd), or a 1032-nt fragment containing a hygromycin (Hyg) resistance gene coding for a hygromycin-phosphotransferase were inserted into the polylinker of pHAVvec9, and termed pHAVvec9-Bsd, pHAVvec9-green fluorescent protein with Bsd (GFP-Bsd), and pHAVvec9-Hyg, respectively (Figure 1)
Summary
Hepatitis A virus (HAV), an atypical Picornaviridae that causes acute hepatitis in humans, grows poorly in cell culture and in general does not cause cytopathic effect. The purpose of the present study is to investigate the maximum size of additional sequences that the HAV genome can tolerate without loosing infectivity. Hepatitis A virus (HAV), a member of the Picornaviridae family, causes acute hepatitis in humans. 7.5 kb single-stranded positive-sense RNA genome [1], which contains a long open reading frame (ORF) flanked by 5' and 3' end non-translated regions (NTR). The HAV long ORF encodes a polyprotein of approximately 250 kDa that undergoes co- and post-translational processing into smaller structural (VP0, VP3, and VP1-2A) and non-structural (2B, 2C, 3A, 3B, 3C, and 3D) proteins [3,4]. The 2A-2B junction is the primary cleavage site of the HAV polyprotein processed by 3Cpro [9,10]. The VP0 undergoes structural cleavage, and an unknown host cellular protease cleaves the VP1-2A junction [11]
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