Characterization of endonuclease associated with gross passage a strain murine leukemia virus

The retroviral genome has three main coding regions: gag, pol, and env, which code for core structural proteins, reverse transcriptase, and envelope glycoproteins, respectively. The 3'-portion of the pol gene has recently been implicated in providing another enzymatic function which is required for integration of the viral DNA into the host chromosome. This function is proposed to be a retroviral long terminal repeat (LTR) sequence-specific DNA endonuclease. The aim of this dissertation was to search for this enzyme in the virion of a murine type C retrovirus and characterize it with respect to its pro posed function in vivo.

An easily solubilized endodeoxyribonuclease of 45,000 daltons was purified to homogeneity from virion preparations of Gross Passage A strain murine leukemia virus. It is optimally active at pH 6.0 in the presence of either 10 mM MgCl₂ MnCl₂. It has no absolute dependence on ATP and is inhibited by 50% in the presence of 90 mM NaCl. Immunoblot analysis revealed that the purified enzyme is not recognized by specific antiserum prepared against pol-encoded proteins, suggesting that it is likely to be of cellular origin.

The authentic virus encoded endonuclease was detected as a 46,000 dalton virion protein on immunoblots. This protein was found to be extremely insoluble in non-ionic detergent lysates of the virus. Partial purification of the protein was achieved by RNase digestion of insoluble particles of disrupted virus, followed by chromatography through a phsophocenulose column. The partially purified chromatographic fractions contained detectable DNA endonuclease activity, which was more active at pH 7.0 than at pH 6.0, and DNA binding activity. Consistent with the DNA sequence-predicted amino acid sequence, the 46,000 dalton protein was found to be basic in nature. The protein was difficult to isolate because of its tight association with other virion proteins and nucleic acids. These interactions could only be completely disrupted with protein denaturants. Similar properties were found in a p46 peptide produced from a specific pol gene-sequence-carrying expression vector in E. coli. This polypeptide was isolated by urea treatment of bacterial pellets, which were prewashed to be essentially free of the bacterial cell proteins. In an attempt to renature the endonuclease protein, the urea extract was subjected to stepwise dialysis in the presence of DNA substrate. Low levels of endonucleolytic activity could be demonstrated with the renatured protein.

These findings suggest that the retroviral pol gene encoded p46 protein is tightly bound within the nucleic acid protein complex of the virion core. The low levels of demonstrable endonuclease activity implies that the enzyme might need other factors to be optimally active in the process of proviral DNA integration into the cellular genome.