Effects of CD81 downregulation by shRNA on viral production, HIV infectivity and Gag localization. (A) Virus release was determined by measuring RT activity in the supernatant of both control and CD81 silenced MOLT/HIV-1 cells. It is expressed as a percentage of the control. Transduction of MOLT/HIV-1 cells with a CD81 shRNA led to an inhibition of HIV-1 production up to 70% (3-fold). (B) Immunoblots showing intracellular CD81 silencing and its effect on viral particle release. MOLT/HIV-1 cells were transduced by HIV-1 based lentivectors containing a shRNA against CD81 or a control shRNA. Three days later, the cells were washed, and resuspended in new medium for 6 hours to allow HIV-1 virion accumulation. The resulting viral particles were run on a SDS-PAGE gel and immunoblotted with an anti-CAp24 antibody to reveal virus particle release. The cells treated with the control shRNA (lane, "sh control") or with the anti-CD81 shRNA (lane "sh CD81") were lysed and total cell protein content were deposited on SDS-PAGE. Resulting immunoblots were probed with different antibodies as indicated. (C) Infectivity of virions issued from shRNA control or CD81 silenced MOLT/HIV-1. The same amount of virus was innoculated on SupT1 cells, and the resulting RT activity from de novo produced virions was detected (See Materials and Methods). The infectivity obtained from the shRNA control virus was referred as 100%. (D) Gag localization at the cell surface by immunofluorescence microscopy. After treatment with lentiviral vectors expressing shCD81 or control shRNA, MOLT/HIV-1 cells were fixed, permeabilized and stained for Gag (using anti-MAp17 antibody) as described in Materials and Methods. Two major phenotypes of Gag were observed: "clustered" – Gag is located in a cluster at one side of the cell surface; or "dispersed" – Gag is distributed all over the cell periphery as punctuated small dots. Patterns were quantified for CD81(-) cells and for the control cells; the numbers were reported on the chart.