Strict Conservation of the Retroviral Nucleocapsid Protein Zinc Finger Is Strongly Influenced by Its Role in Viral Infection Processes: Characterization of HIV-1 Particles Containing Mutant Nucleocapsid Zinc-Coordinating Sequences

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X 2 -Cys-X 4 -His-X 4 -Cys-) designated retroviral (CCHC) Zn 2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn 2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn 2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn 2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn 2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed ∼5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/β-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn 2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn 2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn 2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.