The strict conservation of the HIV-1 retroviral nucleocapsid protein "CCHC" ZN2+-finger is due to its function in early infection events and not RNA packaging

The HIV-1 nucleocapsid (NC) protein contains two highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. To investigate the biological effects of changing ligand binding residues while maintaining Zn2+ coordination, specific mutations were introduced into full length HIV-1 proviral clones. Plasmids containing combinations of NC Zn2+-fingers with His to Cys (CCCC), Cys to His (CCHH), and wild-type fingers were constructed and transfected. Mutant and wild-type particles contained comparable levels of functional viral proteins. Cys-49-His and His-23-Cys mutants packaged wild-type levels of full length genomic RNA whereas all other mutants packaged <50% wild-type levels. All mutants were noninfectious except virions with mutated second position NC fingers. These had titers that were 2 to 3 logs lower than wild-type virus which had a titer of 10(4) TCID/ml. The block in infection in the Cys-28-His/Cys-49-His and His-23-Cys mutants is at the level of integration. These two mutants are able to make full length viral DNA, indicated by the ability to detect two-long terminal repeat (2-LTR) circular DNA forms in Hirt fractionated DNA isolated from infected cells. No 2-LTR products could be detected from the remaining noninfectious mutants indicating a possible block at the level of reverse transcription. The conservation of retroviral NC CCHC Zn2+-fingers is required for early infection processes. The evolutionary pressure to maintain a CCHC Zn2+-finger appears to be due more to its role in early infection processes than its role in genomic RNA packaging.

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