Maturation is an essential step in the life-cycle of human immunodeficiency virus type 1 (HIV-1). It is the transition of the immature, non-infectious virus particle to the mature and infectious virion and is triggered by the proteolytic cleavage of the precursor Gag (Pr55Gag) and GagPol (Pr160GagPol) polyproteins by the viral enzyme protease. Gag is cleaved into the structural proteins matrix (MA, p17), capsid (CA, p24) and nucleocapsid (NC, p7), p6 and two small spacer peptides (p1 and p2). This protease-mediated cleavage elicits the structural rearrangement that results in the dense conical core, characteristic of infectious HIV-1 particles.
Since immature particles are non-infectious, particle maturation is an excellent target for antiretroviral drugs. Protease inhibitors (PI) successfully inhibit viral replication by targeting the enzyme responsible for maturation and have played a major role in antiviral therapy since their introduction in 1995. So far, nine different PIs have been approved for clinical use. However, a high degree of cross-resistance between protease inhibitors limits the utility of these inhibitors if PI resistance emerges.
Maturation inhibitors are a new class of antiretrovirals that also impede particle maturation but do so by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Therefore, direct cross-resistance between PIs and maturation inhibitors may seem unlikely. However during PI treatment, co-evolution of the viral protease and its substrate Gag is common, which may have an effect on the subsequent utility of maturation inhibitors [1–5]. Several maturation inhibitors are or have been in development including: bevirimat (BVM, Panacos PA-457, Myriad MPC-4326); PA1050040, which is a second generation maturation inhibitor from Panacos , based on bevirimat; two maturation inhibitors from Myriad Pharmaceuticals, Vivecon (MPC-9055)[7, 8] and MPI-461359 ; PF-46396  from Pfizer and several capsid assembly inhibitors including CAP-1 , CAI, and BI-257, BI-627 and BI-720 from Boehringer-Ingelheim. Bevirimat was the first of these maturation inhibitors to go into clinical trials and inhibits HIV-1 replication by specifically blocking cleavage of CA from p2, one of the final (rate-limiting) steps in the Gag processing cascade. Incomplete processing of CA from CA-p2 (p25) results in unsuccessful particle maturation and, therefore, non-infectious virions . The CA/p2 cleavage site (CS) has been identified as the bevirimat target region by Western-blotting and in vitro resistance selection studies [14, 15]. Nonetheless, the mechanism of action of bevirimat is still poorly understood as the actual binding site of bevirimat has not been identified. Recently, it has been shown that, besides sterically blocking the CA/p2 junction, bevirimat may have a stabilizing effect on the immature Gag lattice. This indicates that bevirimat binds during assembly and must be incorporated to inhibit maturation, which offers an explanation why bevirimat is unable to prevent cleavage of free Gag in solution.
Initial in vitro selection studies identified bevirimat resistance mutations in the CA/p2 cleavage site at Gag positions 358, 363, 364 and 366 . Phase 2b clinical studies demonstrated that baseline polymorphisms slightly downstream of the CA/p2 cleavage site (Gag aa 369, 370 and 371, known as the QVT-motif) also confer resistance [17, 18].
We previously showed that bevirimat resistance mutations are more frequently observed in PI resistant but bevirimat naïve HIV-1 isolates, compared to PI and bevirimat treatment naïve isolates; and this was mainly attributed to an accumulation of mutations in the QVT-motif . This study also showed that mutations associated with bevirimat resistance were detected more frequently in HIV-1 isolates with three or more PI resistance mutations than in those with less than three PI resistance mutations. Conversely, Adamson and colleagues suggested that mutations in the viral protease affecting viral replication may delay the selection of maturation inhibitor resistance .
To better understand the effect of PI therapy on viral susceptibility to maturation inhibitors, we set up a maturation inhibitor model system. We performed multiple in vitro selection studies with ten different viruses that contained PI resistance mutations in the viral protease and/or Gag CS and that displayed a broad range of replication capacities (RC). Subsequently, we conducted a detailed analysis of the identified resistance mutations. The data in this paper clearly demonstrate that PI resistance mutations alter the resistance profiles for the maturation inhibitor bevirimat. We also show that the protease background determines the level of maturation inhibitor resistance and viral replication.