E levels (RT p66 and integrase) appeared unaltered in virus treated
E levels (RT p66 and integrase) appeared unaltered in virus treated with 20 M AdOx compared with untreated virus samples (Fig. 2A, CEM derived virus). The viral enzyme levels appeared unaltered in AdOx-treated or control HEK293T cell derived virus in similar Western blots (data not shown). However, a small accumulation of full length or partially processed Gag-Pol precursor was consistently observed in 5 independent CEM-derived and 3 HEK293T-derived virus stocks using either a human anti-HIV immunoglobulin (HIV-Ig) (Fig 2A) or a monoclonal antibody specific for Gag (Fig. 2B).Finally, Western blot analysis was performed using HIV-Ig or a goat anti-HIV-1 polyclonal antibody to confirm that the 100 to 170 kDa proteins present in the virion lysates obtained from infected CEM cells were not Env (Fig. 2C). Therefore, whole cell lysates were prepared from Chinese hamster ovary (CHO) cells or CHO cells stably expressing high levels of NL4.3 Env. The lysates were probed by Western analysis using procedures identical to those used for the virion lysates (Fig 2A). It was noted that the HIV-Ig antibody did not detect Env under these conditions (Fig. 2C). Taken together, these results show that AdOx treatment has small effects on Gag-Pol processing resulting in increased amounts of Gag-Pol in virus particles.The virus ultrastructure is altered in the presence of AdOx To further assess what effect AdOx treatment has PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 on virus structure, a cell pellet of day 8 infected CEM cells grown in the presence or absence of 20 M AdOx for 48 hours was thin-sectioned, epon embedded and assessed by transmission electron microscopy (TEM) (Fig. 3). TEM showed numerous virions in each sample and typical viral assembly structures were observed in both control (Fig. 3A, top panel) and AdOx-treated cells (Fig. 3A, bottom panel). In all sections examined, virus production was only observed in morphologically normal cells. Using the maximum diameter, 191 control virions were measured which had a diameter of 103 ?13.8 nm, with a characteristic electron dense viral core structure (Fig. 3B and 3C). We measured 223 virions produced by AdOx-treated CEM cells whichPage 3 of(page number not for citation purposes)Retrovirology 2006, 3:http://www.retrovirology.com/content/3/1/ACEM25 120 100 80 15 60 10 viable cells 5 viability 40 20 0 0 5 10Bviable cells 10E5/mlviabilityHEK293T AdOx M: 0 10 kD 72 55 40 33 24AdOx MAdOx MCEMC10CApRTx10 150HEK293TCApRT x998 7ng/ml66 55 44 33 22 110 5 10ng/ml5 75503 200 10 20AdOx MAdOx MCAp24 ng/mg Total protein9CAp24 ng/mg Total proteinD1015.0 12.5 10.0 7.5 5.0 2.57 6 5 50 4200 5 10AdOx MAdOx ME120CEMCApRTF21CEMRatio CAp24:RTrecovery17 15 13 11 9 7 5 0 5 10 1580 60 40 20 0 0 5 10AdOx MAdOx N-hexanoic-Try-Ile-(6)-amino hexanoic amide site MFigure 1 The effect of AdOx PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 on HIV-1 production in CEM T-cells and HEK293T cells The effect of AdOx on HIV-1 production in CEM T-cells and HEK293T cells. A) CEM cell confluence and viability was assessed using a trypan blue exclusion assay and counting with a hemocytometer. The cell confluence and viability measured in two independent assays and the standard deviation of the mean are shown. B) Western blot of cell lysates from transfected HEK293T cells with the anti-asymmetric dimethylarginine antibody ASYM24 demonstrating a clear reduction in amounts of methylated proteins present in cells treated with 10 M AdOx compared with cells not treated with AdOx. C) Culture supernatant from the treated CEM cells were assayed for CAp24 and RT content. Shown are representa.