Stable APOBEC3H inhibits HIV-1 replication in primary T lymphocytes and inflicts GA-to-AA hypermutations.

<p>A) HIV-1 replication kinetics of the hyper-, lab-, and hypo-Vif variants in CD4+ T lymphocytes from a representative healthy donor encoding unstable A3H haplotype I/I (donor 2). Data from additional experiments using independent donors are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761.s003" target="_blank">Figure S3</a>. Y-axis values represent p24 levels measured by ELISA. B) HIV-1 replication kinetics of the hyper-, lab-, and hypo-Vif variants in CD4+ T lymphocytes from a representative healthy donor encoding one allele of stable A3H haplotype II (and one allele of unstable A3H haplotype I; donor 4). Data from additional experiments using independent donors are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004761#pgen.1004761.s003" target="_blank">Figure S3</a>. Y-axis values represent p24 levels measured by ELISA. C) Immunoblots of the indicated proteins in non-normalized virus-like particle (VLP) supernatants and in cells from days 3, 5, 7, and 9 of the spreading experiments shown in panels A & B. A quantification of the ratio of A3H to CA is shown below each VLP blot set. D) 3D-PCR amplicons generated from proviral DNA of the indicated viruses isolated on day 15 of a spreading infection of unstable A3H (haplotypes I/I and III/IV) or stable A3H (haplotype I/II) donor cells (donors 2, 4, 12). This experiment is representative and performed independently of those shown in panels A-C. E) Histograms depicting the frequencies of GA-to-AA and GG-to-AG mutations under the indicated spreading infection conditions (complementary to the experiment shown in panel D with all sequences derived from independent 98°C high-fidelity PCR amplifications). A minimum of 10 clones were sequenced for each condition (≥5 kb).