Required for the initiation of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27321907 autophagy. Autophagosome biogenesis is coordinated by the phosphatidylinositol 3-kinase (PIK3) complex containing the PIK3 p100 subunit (PIK3C3) and the PI3K p150 subunit (PIK3R4), which associate with Beclin1. WIPI1 regulates the transport of phosphatidylinositol-3-phosphate to the membranes. Two ubiquitin-like conjugation complexes are required; the first one forms a complex between Atg5 and Atg12, and Atg16 is non-covalently bound to this complex in a process that is catalyzed by Atg7 and Atg10. In the second conjugation system, LC3 is cleaved by Atg4 cystein proteases, essentially Atg4A and Atg4B, making it possible for Atg7 and Atg3 to generate the phosphatidylethanolamine (PE)-bound form of LC3: LC3-II. We show that HIV-1 replication can be delayed in stable ATG knockdown cell lines. An additive inhibitory effect was observed when two ATGs were knocked down simultaneously, thus stressing the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29069523 therapeutic potential of this strategy. Importantly, this HIV-1 replication delay was not accompanied by RNAi-induced cytotoxicity, suggesting that autophagy can be targeted in host cells without serious side effects.ResultsStable knockdown of ATG proteins inhibits HIV-1 replicationTo test whether stable knockdown of individual autophagy factors has an effect on HIV-1 replication, we generated cell lines expressing a single shRNA against a specific mRNA encoding one of the 12 autophagy factors (ATGs). Per autophagy factor 4 or 5 shRNAs were tested, resulting in 61 cell lines including 2 controls. The controls were the empty lentiviral vector SHC1 and the vector encoding a scrambled shRNA without a known mRNA target (SHC2). Testing of multiple shRNAs per ATG has several advantages. First, it allows one to score a similar phenotype for different shRNAs that target the same factor, which helps to determine whether the effect is specific. Second, as different shRNAs provide different knockdown efficiencies, the chance that at least one shRNA induces a sufficient knockdown of the specific target increases. To increase the chance of observing antiviral activity, we used a relatively high multiplicity of infection (MOI) of theEekels et al. Virology Journal 2012, 9:69 http://www.Pan-RAS-IN-1 price virologyj.com/content/9/1/Page 3 oflentiviral vectors. Because no shRNAs were available against LC3, we included shRNAs against GABARAPL1, a paralogue of LC3 that has been shown to function in autophagy . All cell lines were tested for inhibition of HIV-1 replication in three independent experiments performed in duplicate. Stable cells were challenged with HIV-1 and the accumulation of CA-p24 in the culture supernatant was monitored. We measured the average CA-p24 concentration at peak infection, corresponding to 10 days post infection. In 14 cell lines at least a log decrease in CA-p24 levels and thus virus replication was observed. Inhibition was measured for the shRNAs ULK1-1 and 4, WIPI1-1 and 3, Beclin1-3, PIK3R4-3, GABARAPL1-3, Atg3-3, Atg4A-1 and 3, Atg5-4, Atg10-3 and 5 and Atg12-4 (Figure 2). To confirm these results, cells freshly transduced with the suppressive shRNA cassettes were again challenged with HIV-1. Virus replication was monitored over a period of 11 days by CA-p24 measurement in the culture supernatant. Inhibition was confirmed for the shRNAs WIPI1-1 and Beclin1-3 (Figure 3A) and in an independent experiment for PIK3R4-3, Atg4A-1 and 3, Atg5-4, Atg10-3 and 5 and Atg16-4 (Figure 3B), but HIV-1 inhibition could not be co.