viral entry - (Jun/13/2011 )

hi friends
is there anyone working with hiv virus and can let me tell the procedure to check viral entry.
my experiment is i infect the macrophage cell with some lysates and then infect with HIV virus .
how does the lystaes helping or inhibiting the viral entry.
i have to use macrophage i cant use any reporter based assay.
i heard that we can check the viral entry by checking p24 but not sure of exact protocol.
it would be of much help if someone can help me come out of this.
Beny

I am not sure what you mean by checking viral entry. You mean really checking the entry of the virus on itself or just check if there is an infection or not?

because the first is done by microscopy... But not sure this is what you need, reading you post it seems not the case.


Check the paper for more information.

thanks for the reply
i just want to check or normalize by result with the entry of virus.
whether entry is same or not in both the cases.........control and test.
how can i do it in the macrophage cell line.

Cant you just check retroviral integration by PCR? and then normalize your data by doing PCR on a cellular gene, like GAPDH?

we make virus by proviral genome transfection in 293T cells. how can we be sure that the RT reaction is due to viral genome only and not whatever DNA we are using for transfection .DNAse may not be 100% effective .how can we rule out that.
is there any other way except real time as it becomes difficult to routinely check real time in our institute.
BENY

Hmmmm......not really sure what you making virus in another cell line (293T) has to do with performing standard DNA PCR on your infected macrophages. If I understand correctly, you make your virus by transfecting your DNA plasmid into a packaging cell line (293T) to make virus, then you use the viral containing supernatant to infect macrophages after you have pre-incubated them with a cell lysate. To me I do not see the problem with doing standard PCR on HIV that has integrated its DNA into the macrophages. Otherwise, you make be able to look at cell cycle arrest since HIV induces a G2/M arrest.........you can do this by flow cytometry after incorporation of a fluorescent marker in your DNA.

chabraha on Fri Jun 24 14:22:08 2011 said:

thanks a lot for replying
ya i am transfecting and making virus in packaging cell line and use the supernatant to infect macrophages.
For which gene we should do the PCR is it necessary to to real time pcr or any general pcr can i do .
real time is not possible for me to do for every sample...........machine is too busy in my institute.
in case of FACS can i quantify it in terms of infection % in different test samples

you can do general PCR (not real-time. If it is important for you to see differences in viral entry between treated and untreated samples you will need to optimize the number of PCR cycles you run to get within the linear range of your primers........as for which primers you use, you would want to make sure that the sequence you choose is highly conserved (maybe the 5'UTR). You also want your amplicon to be small (between 50 & 150bp) to increase specificity. So, if I were you I would just perform a whole genomic DNA extraction at a time when you know the virus has integrated and just PCR for a viral region and a cellular region to normalize your input DNA to.

As for FACS, you wouldn't need to sort the cells, just do flow cytometry to measure DNA amount.........the HIV protein VPR induces G2/M arrest, so you would need to wait until viral proteins are expressed before you check for a G2/M arrest. Nocadazole treatment of your cells should give you an idea what a G2/M arrest looks like by flow cytometry.

Thnaks for the reply.Can you give me any reference if possible.... paper link.