Background
Structural studies have shown that the HIV-1 matrix (MA) protein assembles into hexamers of trimers beneath the viral membrane and undergoes a conformational rearrangement during virion maturation. A recent structural study suggested that this transition is triggered by binding of spacer peptide 2 (SP2), released during protease (PR)-mediated Gag processing, to MA. Although SP2-triggered MA maturation has been linked to Env-mediated membrane fusion, earlier studies reported only minor effects on infectivity when SP2 was deleted, or its cleavage blocked. We also recently reported that the MA-L20K/E73K/A82T mutant replicates with WT kinetics in multiple T-cell lines despite lacking an organized mature MA lattice, leaving the physiological role of MA maturation unclear.
Methods
We developed a liposome-based assay showing that SP2 addition converts liposome-bound MA from the immature to the mature MA lattice. To investigate the role of SP2 in virus replication, we introduced SP2 mutations without altering the overlapping p6* region. We evaluated the impact of SP2 mutations on replication kinetics, infectivity, and fusion kinetics.
Results
Mutations at residues 1, 2, 8, and 13–16 severely impaired infectivity and replication. Long-term passaging of these mutants selected compensatory mutations in SP2, MA, and PR. Most N-terminal mutants, which are expected to be embedded in the viral membrane, reduced fusogenicity but were not rescued by deletion of the Env cytoplasmic tail (CT), unlike PR-deficient virus (PR-D25N). By contrast, mutations at positions 13 and 15 in the C-terminus, which interact with the mature MA lattice, altered fusogenicity and fusion kinetics, respectively, in an Env CT-dependent manner. Notably, these C-terminal mutations also impaired SP2-triggered MA maturation in our liposome assay.
Conclusions
Our results suggest that SP2-triggered MA maturation is not essential for HIV-1 infection but plays a regulatory role in virus-cell fusion. Moreover, SP2 appears to exert functions both related to, and independent of, MA lattice maturation.