Mutations within four distinct Gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleot ide (nt) positions +240 to +274 are thought to form a stem-loop secondary s tructure, termed SL1, that serves as a dimerization initiation site for vir al genomic RNA, We have generated two distinct deletion mutations within th is region, termed BH10-LD3 and BH10-LD4 involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, lon g-term culture of each of these viruses in MT-2 cells resulted in a restora tion of infectiousness, due to a series of compensatory point mutations wit hin four distinct proteins that are normally cleaved from the Gag precursor . In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC , and they involved changes of amino acid Val-35 to Ile within the matrix p rotein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these m utations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. T he results of site-directed mutagenesis studies confirmed that each of thes e four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, p layed the most important role in this regard. Three point mutations, MP2, M NC, and MA2, were also shown to be sequentially acquired by viruses that ha d emerged in culture from the BH10-LD4 deletion. The first two of these wer e identical to those described above, while the last involved a change of V al-35 to Leu. All three of these substitutions were necessary to restore th e infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was abl e to confer some viability on BH10-LD4 viruses. Studies of viral RNA packag ing showed that the BH10-LD4 deletion only marginally impaired encapsidatio n while the BH10-LD3 deletion caused a severe deficit in this regard.