MHIV-mIN Is Infectious

<div><p>(A) Single-cycle infectivity of MHIV-mIN. VSV-G-pseudotyped HIV and MHIV-mIN were made by transfection of 293T cells with plasmid DNA. Infectivity was measured with the MAGI assay by counting β-galactosidase positive cells 2 d post-infection. Virus titers were normalized by the amount of p24 (μg). Infections were performed in triplicate. Mean values are shown here with standard derivation. The background in the assay is about 10 blue cells.</p><p>(B) Copy numbers of late products of reverse transcription were measured by using real-time quantitative PCR. Viral cDNA numbers were normalized by p24. Infections were performed in triplicate. This is a representative experiment of three independent trials.</p><p>(C) Infectivity of MHIV-mIN and MHIV-mIN/m<i>att</i> was compared in the MAGI assay. VSV-G-pseudotyped viruses were prepared and concentrated at 100-fold by ultracentrifugation. Infections of MAGI cells were also performed in the presence or absence of reverse transcriptase inhibitors (50 μM 3TC and AZT). Formation of blue cells in this assay must result from retrovirus infection since addition of reverse transcription inhibitors (shown as black bars) eliminated most of positive cells.</p><p>(D) Comparison of infectivity between wild-type HIV-1 and MHIV-mIN as judged by the ability to make puromycin-resistant clones. Infectivity was measured by counting puromycin-resistant colonies 14 d after infection. To facilitate stable transduction of HIV genomes, a mutation was introduced into the <i>vpr</i> gene in these proviruses because expression of Vpr would preclude formation of colonies due to its cytotoxicity [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-b069" target="_blank">69</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-b077" target="_blank">77</a>]</p>