Expression and characterization of a recombinant novel reverse transcriptase of a porcine endogenous retrovirus.
The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans. Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B. The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs. The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs. However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn(+2) versus Mg(+2)) in the DNA polymerase reactions. As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions. Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.
Avidan
Orna
O
Department of Cell Biology and Histology, Sackler School of Medicine, Tel-Aviv University, Israel.
Loya
Shoshana
S
Tönjes
Ralf R
RR
Sevilya
Ziv
Z
Hizi
Amnon
A
D000595:Amino Acid Sequence; D000818:Animals; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D020077:Endogenous Retroviruses / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D021122:Protein Subunits; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013552:Swine / Q000821:virology*
D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H