HIV-2 and MTT assay, cells lines to use - (Jun/22/2007 )

Hello everyone,

I am doing transfections with an HIV-2 plasmid, made from HIV-2ROD. I don't know if this was successful, I am trying to use the MTT assay to titrate the virus. All wells are positive including the one with undiluted virus stock. I used CBL20 culture supernatant as a positive control, the same result was observed. I concluded that HIV-2 ROD and CBL20 might not infect MT-4 cells. I found out that CBL 20 is an R5 virus, so I got the MT4-CCR5 high cell line. I had the same results.

I am not sure what the problem is, do you know if CEM cells or U87 CCR5 cells work in the MTT assay. Can you use only suspension cells?

Also do you know of cells in which one can generate high stocks of HIV-2?

Thanks,
Olabelle

If you have a plasmid, you should pretty easy get high titer stocks by transfecting 293-cells. (This works really well for HIV-1). For propagation of HIV-2, most cell lines will not work as most lack CCR5 expression or sufficient expression of the other co-receptors used by HIV-2 (HIV-2 can use a lot more than just CXCR4 or CCR5). You could try propagation on U87.CD4.CCR5-cells, but I haven't tried MTT assay on this cell line. Don't you have the possiblity to do an ELISA or RT-activity? Or can you use the GHOST cells which have an LTR-driver EGFP? (they come with different co-receptors, check www.aidsreagent.org).
CEM cells should work in MTT assay, I'm not sure if they are able to support robust replication of HIV-2 (worked with standard cem and hardly got replication of HIV-1, with CEMx174 got really nice replication on the other hand, and there's also CEM-SS cells, but I havent' worked with these, more info found on www.aidsreagent.org).