Molecular Biology International

Review Article

The Continuing Evolution of HIV-1 Therapy: Identification and Development of Novel Antiretroviral Agents Targeting Viral and Cellular Targets

Table 1

Anti-HIV-1 screening assays.


Replication event	Assay	Method

Virus Attachment	HeLa-CD4-LT4-β-gal cells	HeLa-cell based assay measuring reduction in chemiluminescence of HIV-1-infected cells [16]
	TZM-bl cells
	gp120/CD4 Ab binding inhibition	Cell based HIV-1 neutralization assay
	gp120 : CD4 ELISA	Biochemical assay with soluble CD4 and monoclonal gp120 antibodies [17]
	gp120/CD4/coreceptor	Cell based, temperature sensitive fusion assay

Fusion and Chemokine Coreceptor Interaction	HL2/3 cells + HeLa-CD4-LTR-β-gal cells	Cell based assay measuring reduction in chemiluminesence [16]
	Coreceptor inhibiton	GHOST-cell based assays measuring reduction in virus replication
	Coreceptor typing	PBMC and Macrophage cell-based assays with tropism-specific clinical HIV-1 isolates [18]
	Compound displacement of chemokine ligands
	Ca⁺⁺flux

Reverse Transcription	Homopolymer and heteropolymer RT inhibition	Biochemical assay measuring reduction in dGTP-[P32] incorporation [19]
	E/intermediate/late RT products	PCR amplification
	RNaseH inhibition	Biochemical assay [20]
	RT inhibition assays using enzymes with specific mutations	Biochemical dGTP-[P32] incorporation assay [19]

Nuclear localization	2 LTR product in cell nucleus	PCR detection

Integration	Provirus in genomic DNA	PCR detection [21]
	Integrase Complementation	Cell based IN-mutant and Vpr-IN transfection [22]
	Integrase inhibition	Biochemical SPA assay [23]
	Integrase negative virus

Protein Expression	Northern, Western and flow cytometry	Cell based assays with molecular biology endpoints [24, 25]
	Tat, Rev, and Nef inhibition	Biochemical assays [26, 27]
	Cell-based reporter assay for Rev and Tat function
	Intracellular p24	CEM-SS cells infected with HIV-1 and lysed to quantitate p24 by ELISA
	LTR-mediated transcriptional activation

Protease	Intracellular and virion protein processing	Cell based assay with Western analysis [28]
Protease	Polyprotein cleavage	Biochemical FRET assay [29]