Polymorphisms in the CypA-binding loop of the viral capsid modulate HIV-1 susceptibility to MxB inhibition. (A) Illustration of the major polymorphisms in the CypA-binding loop of HIV-1 capsid. The prevalence of each variant was calculated on the basis of the capsid sequences (5372 entries) that are available at HIV database (http://www.hiv.lanl.gov/content/index). The position of CypA-binding loop is shown in the context of capsid structure (adapted from PDB ID 3P05) [19,20]. The capsid sequence from amino acid positions 85 to 96 is from the HIV-1NL4–3 strain. (B) Each of the nine major polymorphic amino acids was inserted into HIV-1NL4–3. The wild type NL4-3 and mutated viral DNA were transfected into HEK293T cells to produce progeny virus particles. Virus amounts were determined by measuring viral RT activity. Viruses of equal RT levels were used to infect the TZM-bl indicator cells. Results are summarized in the bar graph with the infectivity of wild type NL4-3 set at 1. (C) Wild type NL4-3 and its mutants were used to infect MxB-expressing SupT1 cells. Folds of inhibition by MxB were calculated as described in Figure 1A. (D) Incorporation of CypA into the wild type and mutated HIV-1NL4–3. Viral DNA was transfected into HEK293T cells. Virus particles in the culture supernatants were harvested by ultracentrifugation through a sucrose gradient as described in . Virus amounts were determined by HIV-1 p24 ELISA. Virus particles of the same p24 quantities were examined in Western blotting for the presence of CypA. Lysates of transfected HEK293T cells were also subject to Western blotting to assess levels of endogenous CypA and viral Gag/p24 expression.