Barbour, N
Barbour, N. replication efficiency and should show useful for the evaluation of patient samples in Mirk-IN-1 clinical trials. Replication fitness can be defined as the efficiency with which a computer virus replicates in response to the selective pressures present in its environment. Human immunodeficiency computer virus type 1 (HIV-1) replication fitness is usually postulated to influence which variants predominate in an HIV-infected patient’s quasispecies and to impact treatment responses and disease progression (28, 31, 39). A critical question is usually whether assays that measure HIV-1 replication efficiency in cell culture are affordable surrogates of viral replication fitness in patients and thus can be used to predict prognosis of HIV-1 contamination or response to therapy. If so, assays of HIV-1 replication efficiency might be used to determine how aggressively to initiate treatment and the optimal time to switch a failing regimen. All assays that measure HIV-1 replication efficiency in cell culture compare the clinical (or test) computer virus to a reference HIV-1 isolate. These assays vary widely, although they Mirk-IN-1 differ primarily in at least one of four different characteristics. First, such assays may either measure the replication efficiency of computer virus cultured directly from the patient sample or a recombinant computer virus constructed by combining a PCR-amplified segment of the patient virus genome with the sequence backbone from a laboratory strain of HIV-1. Second, these assays may measure the replication efficiency during a single virus replication cycle or over multiple cycles. Third, these assays may measure computer virus growth directly, using a viral gene or gene product, or measure computer virus growth indirectly by assaying a reporter gene substituted for any nonessential viral gene. Fourth, assays of HIV-1 replication efficiency may evaluate the growth of test and reference viruses in the same or individual cultures (referred to as growth competition and parallel infections, respectively) (28, 31). Growth competition assays are considered to be the preferred method for measuring HIV-1 Mirk-IN-1 replication fitness in cell culture because they are more sensitive to subtle differences in replication efficiency than parallel infections and are not subject to artifact due to differences in culture conditions CDC18L (10, 30). Growth competition assays require a method to distinguish the two computer virus variants, usually by measuring the relative proportions of each mutant (or a linked reporter gene) using bulk DNA sequencing, clonal analysis, heteroduplex tracking assay, or real-time PCR (16, 22, 26, 27, 32, 33, 36, 40). These methods make growth competition assays more labor-intensive than parallel infections, thus limiting their use in larger clinical trials. We have designed a multiple-cycle, recombinant-virus, growth competition assay that obviates the need to purify and analyze HIV-1 RNA or DNA. Two recombinant HIV-1 computer virus constructs were designed to express either the mouse or the mouse genes in place of HIV-1 gene in place of (29, 35). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat Thy1.1 (HIS51) and R-phycoerythrin (PE)-conjugated rat anti-mouse Thy1.2 (30-H12) monoclonal antibodies were obtained from BD Pharmingen (San Jose, CA). The PM1 cell collection, a clonal derivative of HUT 78 that is permissive for both macrophage-tropic and lymphocyte-tropic strains of HIV-1, was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Marvin Reitz (24). The 293 cell collection was obtained from American Type Culture Collection (ATCC, Rockville, MD). Fetal bovine serum (FBS) was Mirk-IN-1 obtained from Valley Biomedical (Winchester, VA) or the ATCC. Restriction enzymes were obtained from either New England Biolabs (Beverly, MA) or MBI Fermentas (Hanover, MD). Cell culture. PM1 cells were grown in the presence of RPMI Mirk-IN-1 (Cellgro, Herndon, VA), supplemented with 10% FBS, l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 U/ml). 293 cells were produced in Dulbecco altered Eagle medium with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml). Construction of pAT2 and pAT1 HIV-1 vectors. Construction of the pAT1 and pAT2 vectors shown in Fig. ?Fig.11 was.