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

Abstract

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this 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, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within 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 protein (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 mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.

HIV-1 Pr55Gagbinds genomic and spliced RNAs with different affinity and stoichiometry

“…86 Interestingly, this genomic site generated a consensus over the last years, since Pr55 Gag binding sites in immature virions and NCp7 binding sites in mature virions, 17,69 fit with present findings and our previous in vitro data (Table S1). 85,86 Other studies showed that deletion of the lower part of SL1 101,102 or mutation of its internal loop 32 reduces gRNA packaging and viral infectivity. The effect of mutations in the SL1 internal loop on in vivo gRNA dimerization 32,103-105 is probably a direct consequence of the weaker binding of Pr55 Gag to the mutated SL1, the apical loop of which mediates gRNA dimerization.…”

Role of MxB in Alpha Interferon-Mediated Inhibition of HIV-1 Infection

“…The pVSV-G plasmid was cotransfected to produce HIV-1 particles that carry the VSV glycoprotein. The amounts of HIV-1 produced were determined by either measuring HIV-1 capsid/p24 by enzyme-linked immunosorbent assay or measuring HIV-1 reverse transcriptase activity, as previously described (40). An amount of HIV-1 equivalent to 100 ng p24 was used to infect different cell lines.…”

Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1

“…Mutations in and around the packaging signal region of the HIV-1 genome drive evolution of compensatory mutations in long-term culture [ 22 , 101 , 102 , 103 ]. Intriguingly, these are found in the Gag coding region and seem to act at the protein rather than at the RNA level [ 104 ].…”