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.

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.

Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn(2+) fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn(2+) fingers. The sequence of the NH(2)-terminal NC Zn(2+) finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn(2+) finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn(2+) fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn(2+) finger compared to mutant and wild-type Zn(2+) fingers in NC that support replication. The present study shows that six of seven of the Zn(2+) fingers from the CNBP protein can be used as substitutes for the Zn(2+) finger in the NH(2)-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn(2+) finger mutants.