Eny virions released in its presence (t1/2 = 24.5 h). Data represent normalized
Eny virions released in its presence (t1/2 = 24.5 h). Data represent normalized inhibition to the DMSO control (set at 100 ).Desimmie et al. Retrovirology 2013, 10:57 http://www.retrovirology.com/content/10/1/Page 4 ofactivity and p24 protein in the Mangafodipir (trisodium)MedChemExpress Mangafodipir (trisodium) supernatants at 24 and 72 h post infection (hpi), respectively. Unlike raltegravir and irrespective of the extensive washing, ritonavir and CX05045 profoundly impaired virus replication when added during production (Figure 2B, C), ruling out that the effect is caused by the carry-over of compound in the supernatant. To further corroborate the late effect of LEDGINs on infectivity of HIV-1, we produced single round VSV.G pseudotyped HIV pseudovirus in the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25432023 presence or absence of CX05045 and measured the firefly luciferase (fLuc) activity in MT-4 cells. Addition of CX05045 during production resulted in lower fLuc activity (e.g. >300-fold for the 1:1 dilution) compared to the DMSO-treated virus (Figure 2D). We then examined the replication cycle of HIV in time using qPCR analysis of viral DNA species [18] and time-of-addition (TOA) [19]. Consistent with our previous report on the mode of action of LEDGINs in the early stage of HIV replication [12], CX05045 blocks HIV-1 integration without affecting the upstream replication events (Figure 2E). While only AZT inhibited RT activity, both CX05045 and raltegravir significantly blocked integration resulting in an accumulation of 2long terminal repeat (2-LTR) circles at 24 hpi (Figure 2E), a hallmark of IN inhibitors [12,20,30]. Next, we designed and carried out a TOA experiment in MT-4 cells in which the antivirals were added every hour post infection and the supernatants were harvested 31 hpi, the average duration of a single HIV replication cycle in laboratory-adapted T cells [21,22]. Theoretically, addition of a drug after the completion of the step targeted will result in a lack of inhibition and hence p24 protein will accumulate in the supernatant. As such, the targeted step by CX05045 or the control inhibitors (AZT, raltegravir and ritonavir) was monitored by quantifying p24 protein in the supernatants harvested from the TOA experiment (Figure 2F, Additional file 1: Table S1). The average time delay post infection (t0) when addition of the compound retained 50 inhibition of HIV-1 replication (t1/2) was calculated [23]. Accordingly, we found t1/2 of 7.0, 12.4, 12.1 and 25.7 hpi for AZT, raltegravir, CX05045 and ritonavir, respectively (Figure 2F, Additional file 1: Table S1). These correspond to RT (AZT), integration (raltegravir and the early effect of CX05045) and proteolytic maturation steps (ritonavir). Subsequently, to pinpoint the late effect of LEDGINs, we used the supernatants harvested from the TOA experiment and evaluated the replication capacity of the progeny virions. To do this, we infected new MT-4 cells with the supernatants and quantified p24 protein in the supernatants 4 days post infection (dpi) (Figure 2G, Additional file 1: Table S1). As expected, cells incubated with supernatants harvested from cells treated with AZT (beyond 4 hpi) or raltegravir (beyond 11 hpi) in theTOA experiment displayed comparable productive infection as PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 the control virus (DMSO-treated) infected cells, coinciding with their targets i.e. RT and integration, respectively (Figure 2G). On the other hand, viruses produced in the presence of ritonavir added as late as 21 hpi in the TOA experiment were less infectious, corresponding to the proteolytic matur.

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