The population of human immunodeficiency virus type 1 (HIV-1) present in a single infected patient at any given time can show remarkable diversity. Moreover, the extent of diversity can evolve over time and is different in different genes. The most striking changes in diversity occur in the envelope glycoproteins (Env). The initial transmission of HIV-1 can result in infection of the new host with multiple viruses expressing genetically diverse env sequences [1-6]. Early in the evolution of infection, however, viruses expressing extremely homeogeneous env sequences become dominant, presumably reflecting the selection of viruses that are best adapted for replication in available target cells, and/or resistant to the nascent host immune response [1-3,7]. This initial homogenization is followed by a period often lasting many years, in which both the diversity of the env sequences and the evolutionary distance from the initially dominant strain increase linearly by approximately 1% per year [5,8-17]. Subsequently, the extent of viral diversity begins to plateau and, in the late stages of disease, a decline in viral diversity can be observed [8,11,12,18].
Although genetic diversity of the viral env has been extensively studied, less information is available concerning the extent that these genetically diverse Env proteins also display functional diversity. Envelope sequences have been amplified from plasma or short-term cell cultures and used to produce recombinant or pseudotyped viruses expressing primary env sequences [19-25]. Most studies have found that only 40–70% of such viruses are infectious, but quantitative assessment of the replicative capacity of a large number of viruses expressing different envelope sequences from a single patient has not been reported.
It also remains unclear the extent to which other properties of the viral Env proteins are shared by coexisting quasi-species from a given patient. Viral isolates obtained from different individuals can differ in their sensitivity to inhibition by chemokines [26-30], entry inhibitors [31-37], certain monoclonal antibodies [32,38], and autologous serum [26,39], but the extent that different viruses obtained from the same individual show similar sensitivity to a given entry inhibitor has not been extensively evaluated. Furthermore, replicative capacity, per se, can influence the sensitivity of viruses to inhibitors of entry [26,31,36,40], but it remains unknown whether or not the sensitivity of viruses from a given patient to entry inhibitors correlates closely with replicative capacity.
We have recently described an approach that allows the direct isolation of contemporaneous clonal viruses from the plasma of infected individuals, including viruses capable of using CCR5 and/or CXCR4 viral coreceptors [41,42]. These viruses are potentially useful for the evaluation of the functional correlates of env genetic diversity. First, each clonal virus emerges independently, and therefore viruses with low infectivity are not lost through competition with rapidly replicating viruses. Furthermore, the env sequences expressed by these viruses are genetically diverse, and the functional properties have not been modified by through mutation or recombination occurring during PCR. In this study, we have created recombinant viruses expressing Env proteins from these clonal viruses in a reporter construct expressing luciferase activity, and evaluated: i) the spectrum of infectivity observed for Env proteins expressed by contemporaneous viral clones from the same patient, ii) the ability of these viruses to infect different target cells, and, iii) the relationship between infectivity and the susceptibility of the Env proteins to several different entry inhibitors.