A monocyte-derived factor interferes with detection of reverse transcriptase in HIV-1 infection

Area of Science:

• Virology
• Immunology
• Biochemistry

Background:

• Rabbit macrophage cell line 6083 infected with human immunodeficiency virus type 1 (HIV-1) showed no reverse transcriptase (RT) expression despite productive infection.
• Standard RT assays may be compromised by unknown inhibitory factors in cell culture supernatants.

Purpose of the Study:

• To identify the cause of suppressed RT activity in HIV-1 infected rabbit macrophages.
• To characterize the inhibitory factor interfering with RT assays.

Main Methods:

• Analysis of culture supernatants from infected and uninfected cell lines (6083, U937) and primary human monocytes.
• Characterization of the inhibitory factor's properties (heat lability, molecular weight, sensitivity to chemical modifications).
• Assessing the effect of the inhibitory factor on RT substrates and products and testing potential inhibitors (S-dG30, EGTA).

Main Results:

• A monocyte-derived inhibitory factor (MDIF) was identified in supernatants from uninfected cultures.
• MDIF is a heat-labile protein of approximately 40 kD that degrades RT substrates and products.
• MDIF activity requires divalent cations, with a preference for calcium, and is inhibited by EGTA.
• While S-dG30 inhibits MDIF, it also inhibits RT, making it unsuitable for assay correction.

Conclusions:

• MDIF interferes with standard reverse transcriptase assays, leading to false negatives.
• The presence of MDIF should be considered when interpreting RT levels as a measure of retroviral infection.
• EGTA is recommended for minimizing MDIF interference in RT assays due to its calcium-chelating properties.