Characterization of the block in replication of nucleocapsid protein zinc finger mutants from Moloney murine leukemia virus

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn 2+ fingers (-Cys-X-2-Cys-X-4-His-X-4-Cys- [CCHC]) perform multiple function s in the virus life cycle. Moloney murine leukemia virus mutants His 34-->C ys (CCCC) and Cys 39-->His (CCHH) were able to package their genomes normal ly but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Prim er tRNA placement on the viral genome and the ability of the tRNA to functi on in reverse transcription initiation in vitro also appear normal. Some "f ull-length" DNA copies of the viral genome were synthesized in mutant virus -infected cells. The CCCC and CCHH mutants produced these DNA copies at gre atly reduced levels. Circle junction fragments, amplified from two-long-ter minal-repeat viral DNA (vDNA) by PCR were cloned and characterized. Remarka bly, it was discovered that vDNA isolated from cells infected with mutant v irions had a wide variety of abnormalities at the site at which the two end s of the linear precursor had been ligated to form the circle (i.e., the ju nction between the 5' end of U3 and the 3' end of U5). In some molecules, b ases were missing from regions corresponding to the U3 and U5 linear vDNA t ermini; in others, the viral sequences extended either beyond the U5 sequen ces into the primer-binding site and 5' leader or beyond the U3 sequences i nto the polypurine tract into the env coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defectiv e genomes would certainly be unsuitable substrates for integration. Thus, s trict conservation of the CCHC structure in NC is required for infection ev ents prior to and possibly including integration.