Human Immunodeficiency Virus Type 1 Nucleocapsid Zn 2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA

Abstract

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn 2+ finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn 2+ fingers (NC H23C and NC H44C ), an integrase mutant (IN D116N ), a double mutant (NC H23C /IN D116N ), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn 2+ finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.