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Journal of Virology, October 1999, p. 8185-8195, Vol. 73, No. 10
0022-538X/99/$04.00+0

Characterization of the Block in Replication of Nucleocapsid Protein Zinc Finger Mutants from Moloney Murine Leukemia Virus

Robert J. Gorelick,1,* William Fu,2,dagger Tracy D. Gagliardi,1 William J. Bosche,1 Alan Rein,2 Louis E. Henderson,1 and Larry O. Arthur1

AIDS Vaccine Program, SAIC Frederick,1 and Molecular Virology and Carcinogenesis Laboratory, ABL-Basic Research Program,2 National Cancer Institute Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

Received 15 March 1999/Accepted 2 July 1999

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn2+ fingers (-Cys-X2-Cys-X4-His-X4-Cys- [CCHC]) perform multiple functions in the virus life cycle. Moloney murine leukemia virus mutants His 34right-arrowCys (CCCC) and Cys 39right-arrowHis (CCHH) were able to package their genomes normally but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Primer tRNA placement on the viral genome and the ability of the tRNA to function in reverse transcription initiation in vitro also appear normal. Some "full-length" DNA copies of the viral genome were synthesized in mutant virus-infected cells. The CCCC and CCHH mutants produced these DNA copies at greatly reduced levels. Circle junction fragments, amplified from two-long-terminal-repeat viral DNA (vDNA) by PCR, were cloned and characterized. Remarkably, it was discovered that vDNA isolated from cells infected with mutant virions had a wide variety of abnormalities at the site at which the two ends of the linear precursor had been ligated to form the circle (i.e., the junction between the 5' end of U3 and the 3' end of U5). In some molecules, bases were missing from regions corresponding to the U3 and U5 linear vDNA termini; in others, the viral sequences extended either beyond the U5 sequences into the primer-binding site and 5' leader or beyond the U3 sequences into the polypurine tract into the env coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defective genomes would certainly be unsuitable substrates for integration. Thus, strict conservation of the CCHC structure in NC is required for infection events prior to and possibly including integration.

* Corresponding author. Mailing address: AIDS Vaccine Program, SAIC Frederick, NCI-FCRDC, Bldg. 535, Room 410, P.O. Box B, Frederick, MD 21702-1201. Phone: (301) 846-5980. Fax: (301) 846-7119. E-mail: gorelick{at}avpaxp1.ncifcrf.gov.

dagger Present address: Laboratory of Neurovirology, Institute of Clinical Research, Veterans Affairs Medical Center, Washington, DC 20422.

Journal of Virology, October 1999, p. 8185-8195, Vol. 73, No. 10
0022-538X/99/$04.00+0


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