Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

In vitro infection of SUP-T1 cells with HIV-1 LAI. The design of our experiment comprised treating SUP-T1 cells with HIV-1 strain LAI, UVinactivated HIV-1 strain LAI (HIV UV ), or cell-conditioned medium as mock infection. Experimental steps were performed as described in reference 1 but with the following changes. HIV UV was generated by irradiating HIV preparations for 5 min, a dose we found sufficient to abrogate viral replication in MAGI-CXCR4 cells as detected by X-Gal (5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside) staining. Lack of replication with HIV UV was confirmed in SUP-T1 cells by viral mRNA load by Taq-Man qPCR (35) . Both live virus and UV-inactivated virus were added to 5 ϫ 10 6 cells at a multiplicity of infection of 5. This dose of live virus was found to achieve 100% infection at 24 hpi with~50% cell viability as measured by trypan blue exclusion assay. Mock infections were performed with an identical volume of SUP-T1-conditioned medium. Each treatment was performed in triplicate, and replicates were sampled at 5, 12, and 24 hpi.