Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3'' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.     

Insertion mutants of herpes simplex virus have a duplication of the glycoprotein D gene and express two different forms of glycoprotein D.

We produced insertion mutants of herpes simplex virus (HSV) that contain two functional copies of genes encoding different forms of glycoprotein D (gD). These viruses have the gene for HSV type 2 (HSV-2) gD at the normal locus and the gene for HSV-1 gD inserted into the thymidine kinase locus. Results of immunoprecipitation experiments done with monoclonal antibodies revealed that both gD genes were expressed by these viruses, regardless of orientation of the inserted HSV-1 gD gene, and that maximal synthesis of both glycoproteins depended on viral DNA replication. This apparently normal expression of the inserted HSV-1 gD gene was from a DNA fragment (SacI fragment, 0.906 to 0.924 map units) containing nucleotide sequences extending from approximately 400 base pairs upstream of the 5' end of the gD mRNA to about 200 base pairs upstream of the 3' end. The glycoproteins expressed from both genes were incorporated into the surfaces of infected cells. Electrophoretic analyses of purified virions and neutralization studies suggest that both glycoproteins were also incorporated into virions. This nonpreferential utilization of both gene products makes these viruses ideal strains for the generation and characterization of a variety of mutations.