2000. assessed positive selection pressure on the region in all three patient groups. An increase in positive selection was observed in cleavage site regions p7/p1/p6 only after the acquisition of major PI mutations, suggesting that amino acids in cleavage sites under positive selection pressure could function as compensatory mutations for major PI mutations in the protease region. Isolated mutations did not appear to confer PI resistance, but mutations in the cleavage sites could substitute for minor PI resistance mutations in the protease region. The introduction of highly active antiretroviral therapy (HAART) has substantially improved the prognosis of human immunodeficiency computer virus (HIV)-infected patients (18). However, the effect of HAART has been hindered by the generation of mutations in the viral genome that confer drug resistance. In HAART patients in whom viral replication is usually incompletely suppressed, antiretroviral treatment can lead to selection pressure, resulting in resistance mutations. Specifically, many anti-HIV drugs target the HIV protease and reverse transcriptase polymerase enzymes, so resistance mutations are clustered in these regions. HIV-1 protease inhibitors (PIs) compete with the substrate for the active site of the protease. The current thought is usually that major resistance mutations to PIs alter the configuration of the enzyme’s active site, thereby decreasing the effectiveness of the enzyme and reducing the fitness of the computer virus. However, to compensate for the reduced activity of the enzyme, secondary mutations develop in the protease gene which increase substrate cleavage and thus improve viral fitness (6). An alternative theory has been proposed by Nijhuis et al. (21), Verheyen et al. (28), and Dam et al. (6), who suggest that mutations in the region are involved in the development of resistance against PIs and that primary mutations can make the computer virus resistant to PIs in the absence of any major mutations in Tegoprazan the protease gene (21). Serial passage studies by Aoki et al. (3) indicate that a broad range of mutations are involved in PI resistance. Positive selection is usually a driving pressure in the generation of mutations in the pol region of the HIV genome, and sites under positive selection pressure include many major and minor resistance mutations in the protease region (5). Selective forces working on the region have been described mainly for cytotoxic T-lymphocyte (CTL) responses rather than for antiviral drug responses (10). Two mechanisms may induce mutations in the region during PI treatment. First, the drug can select for specific resistance mutations in region as well as in the protease region. We aimed to test if there is more selection on codon sites in patients harboring PI mutations than in patients without PI mutations. We also performed a longitudinal study with sequential sampling to determine whether the generation of resistance mutations in the region was facilitated by the presence of specific resistance mutations in the protease gene or whether they occurred independently of major protease resistance mutations. MATERIALS AND METHODS Setting. Denmark has a populace of 5.4 million, and the estimated prevalence of HIV contamination in the adult populace is 0.07% (17). Denmark’s tax-funded health care system provides antiretroviral treatment free of charge to all HIV-positive residents. Treatment of HIV contamination is restricted to eight specialized medical centers. Resistance testing is usually centralized in the Hif3a Department of Virology, Statens Serum Institute, Copenhagen, Denmark. Data sources. We extracted clinical data as well as data for antiretroviral treatment from the Danish HIV Cohort Study, Tegoprazan which is described elsewhere (17). In brief, the cohort is usually ongoing and includes all HIV patients seen in the eight Danish HIV clinics since 1 January 1995. In November 2008, the cohort included 5,300 patients. HIV sequences were obtained from the Danish HIV Sequence Database, housed at the Department of Virology, Statens Serum Institute; this included sequences from resistance assessments performed from 2000 to 2008. The database currently includes 6,000 sequences from 2,200 HIV-infected patients. Cross-sectional study populace. In the first part of the study, we identified all Danish HIV-infected patients who were registered in the Danish HIV Cohort Study and had a sequence available in the Danish HIV Sequence Database that had been collected in the period from 1 January 2004 to 31 December 2005, were infected with HIV subtype B, and had complete sequences available for the protease as well as for the regions (spanning the C-terminal end of the gene and made up of the two cleavage sites [CS], p7/p1 and p1/p6). When several sequences were available from a patient, only the first was included in the study. A total of 313 patients fulfilled these criteria. This populace was further divided into three subgroups. Group 1 Tegoprazan (= 160) included HIV-1-infected patients who had never received antiretroviral treatment, i.e., antiretroviral-na?ve patients. Group 2 (= 93) included HIV-1-infected patients treated with a failing HAART.