J Bacteriol Virol.  2006 Sep;36(3):185-194. 10.4167/jbv.2006.36.3.185.

Molecular Characterization of Porcine Endogenous Retrovirus gag Genes from Pigs in Korea

• Lee J
• Lee D
• Yoo JY
• Kim GW
• Park HY
• Lee HT
• Kim YB

• KMID: 2055026
• DOI: http://doi.org/10.4167/jbv.2006.36.3.185

Abstract

Xenotransplantation, as a potential solution to the shortage of human organs, is associated with a number of concerns including immunologic rejection and xenogenic infection. While the pigs are considered the most suitable organ source for xenotransplantation, there is a potential public health risk due to zoonosis. Among the known porcine zoonotic microbes, Porcine Endogenous Retrovirus (PERV) is the most considerable virus. PERV belongs to the Gammaretrovirus and has been divided into three groups (A, B, and C). To characterize the gag of PERVs, we isolated the genomic DNAs from three pig breeds (Birkshire, Duroc, and Yorkshire) and two types of SPF miniature pigs. About 1.5 kb fragments covering full length of gag were amplified and cloned into T-vector. A total of 38 clones were obtained and sequenced. Nucleotide sequences were analyzed and phylogenetic trees were constructed from the nucleotide and deduced amino acids. PERV-A, -B and -C were present in the proportion of 47, 19 and 34%, respectively. Regardless of origin or subgroups, gag clones showed highly homology in nucleotide and deduced amino acid sequences. Deduced amino acids sequence alignments showed typical conserve sequences, Cys-His box and processing sites. Among analyzed clones, about 28% of isolates had the correct open reading frame. To test the functional expression of Gag protein, gag was subcloned into expression vector and confirmed its expression in HeLa cell. This research provides the fundamental information about molecular characteristics of gag gene and functional Gag protein related xenotropic PERVs.

Keyword

PERV; Xenotransplantation; Group specific antigen; Zoonosis

MeSH Terms

Amino Acid Sequence
Amino Acids
Base Sequence
Clone Cells
DNA
Endogenous Retroviruses*
Gammaretrovirus
Gene Products, gag
Genes, gag*
HeLa Cells
Humans
Korea*
Open Reading Frames
Public Health
Sequence Alignment
Swine*
Transplantation, Heterologous
Amino Acids
DNA
Gene Products, gag

Figure

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  Figure 1. Construction of vaccinia virus recombinant. HeLa cells transfeted with a PERV gag plasmid are infeceted with wild type vaccinia virus (WR). Homologous recombinantion occurs between flanking sequences beside gag gene and virus genomic DNA. In a consequence of PERV gag gene insertion into viral DNA, recombinant virus was produced.

• 

  Figure 2. Alignment of deduced amino acids sequences of PERV Gag. Full length nucleotide sequences of gag clones were identified. Deduced amino acids sequences of cloned gag gene clones were aligned with the reference strains (PERV-A; AJ279056, PERV-B; AJ133816, PERV-C; AF038600). Each type of clones had homology in the nucleotide sequences between 241 and 440. The sequences in shadowed boxes indicate the cleavage sites of Gag, in the closed box indicate Cys-His box.

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  Figure 3. Comparison of deduced amino acid sequence of PERV gag clones. PERVs (PERV-A: AJ279056, PERV-B: AJ133816, PERV-C: AF038600), GaLV (Gibbon ape leukemia virus, NC001885), BaEV (Baboon endogenous virus, AF142988), FeLV (Feline leukemia virus, K01803), MoMLV (Moloney murine leukemia virus, AF033811), and MuLV (Murine leukemia virus, AB213652) were used as references for alignment of cloned Gag deduced amino acids sequences. Cys-His motif in shadow region, pivotal in virus packaging, was conserved in alignment all gamma-retroviruses.

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  Figure 4. UPGMA clustering tree based on 1.5 kb nucleotide sequences of PERV gag. The tree was generated by the method of Kimura (1980) on the basis of gag sequences using the Treecon (ver 1.3b.). Numbers at nodes indicate the bootstrap value of 100 resampled datasets. PERV-A (AJ279056 and AJ293656), PERV-B (AJ133816, AJ133818 and AJ279057), PERV-C (AF038599 and AF038600) and PERV-E (AF356698) were used reference strain. Upper scale bar means distances.

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  Figure 5. Immunoblot analysis of expressed PERV Gag protein. Recombinant vaccinia virus harboring PERV gag gene was amplified in HeLa cell. Amplified recombinant vaccinia virus was coinfected with vTF7–3, vaccinia virus producing T7 polymerase, to HeLa cell (lane 1, lane 2). HeLa cell was harvested after 36 hrs later. Detection of expressed PERV Gag protein was performed by anti-His Ab. HeLa cell was used as a control (lane C). The sizes of the molecular weight markers are indicated on the left of panels.

Reference

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