Measurement of Human Immunodeficiency Virus Type 1 Plasma Virus Load Based on Reverse Transcriptase (RT) Activity: Evidence of Variabilities in Levels of Virion‐Associated RT

Abstract

Virus load based on levels of functional reverse transcriptase (RT) was measured in plasma from 50 human immunodeficiency virus (HIV) type 1 -infected persons, in 87 samples from 10 HIV-1 seroconversion panels, and in 100 uninfected persons by use of Amp-RT, an ultrasensitive RT assay. Of the 50 clinical samples, 38 (76%) were Amp-RT positive, while all uninfected controls were negative. Pearson's correlation coefficient of RNA and RT levels was .73 for all samples, .86 for seroconversion samples, and .49 for clinical samples. Calculated ratios of RT activity to virion RNA varied widely during both early and late stages of infection. Mean RT:RNA ratios in 8 seroconversion panels and in 12 (34.3%) of 35 individual clinical samples were significantly lower than the ratio for a reference virus. However, ratios were stable in individual seroconversions over time. These data demonstrate that RT activity can be used to quantitate plasma virus load and provide evidence of different levels of virion-associated RT among HIV-1 -infected persons. inability of RNA-based methods to provide functional and School of Medicine, Kumamoto, Japan. phenotypic information on virus loads illustrates the need

HIV‐1 viral load determination based on reverse transcriptase activity recovered from human plasma

“…As can be extracted from Figure 4 and Table V, the variation in the RT/RNA ratio was 100 fold between individual samples. This is in agreement with a previous study using the Amp-RT assay [Garcia Lerma et al, 1998], where they in addition found that this ratio remained relatively constant for consecutive samples from the same patient (see Highest RT value (757 fg/ml), found in a sample with 5,600 copies/ml, excluded from mean calculation.…”

“…Earlier, we have shown accurate measurement of HIV RT activity in plasma during primary infection before seroconversion [Awad et al, 1997]. The presence of RT activity in the acute stage and after seroconversion was shown with both the Amp-RT and the Product Enhanced RT (PERT) assays, which use PCR amplification of DNA produced by RT [Garcia Lerma et al, 1998Lerma et al, , 2000Bü rgisser et al, 2000]. Interestingly, the correlation between Amp-RT activity/ml and RNA copies/ml, measured by Nucleic Acid Sequence Based Amplification (NASBA), was found to be much stronger in a panel consisting of acute stage plasma compared to the correlation found with a panel of convalescent plasma, even after compensation for antibody-mediated RT inhibition.…”

“…Interestingly, the correlation between Amp-RT activity/ml and RNA copies/ml, measured by Nucleic Acid Sequence Based Amplification (NASBA), was found to be much stronger in a panel consisting of acute stage plasma compared to the correlation found with a panel of convalescent plasma, even after compensation for antibody-mediated RT inhibition. This indicated that RTinhibiting antibodies found after seroconversion played a strong role in the Amp-RT assay [Garcia Lerma et al, 1998]. …”

The Level of Reverse Transcriptase (RT) in Human Immunodeficiency Virus Type 1 Particles Affects Susceptibility to Nonnucleoside RT Inhibitors but Not to Lamivudine

“…Increased enzymatic activity is biochemically equivalent to increased levels of a less active enzyme. Strikingly, HIV-1 isolates have been obtained from NNRTI-naive patients with variations in RT activity per RNA or infectious unit, suggesting that natural variation in RT activity per virion is common (6,10). It will be interesting to determine if NNRTIs can select for an increase in RT activity in HIV-1 isolated from NNRTI-experienced individuals.…”

The Triple Threat of HIV-1 Protease Inhibitors

“…These differences have been attributed mostly to processing defects (Kaplan et al 1993;de la Carriére et al 1999;Müller et al 2009), but reduced RT packaging has also been suggested (Bleiber et al 2001). Importantly, the amount of functional RT in the virion closely correlates with infectivity (García Lerma et al 1998;Marozsan et al 2004) because viruses with reduced RT functionality are less efficient in completing reverse transcription (Bleiber et al 2001;Julias et al 2001;Wang et al 2010). Furthermore, particles with reduced RT content or activity display increased sensitivities to NNRTIs (Ambrose et al 2006;Henderson et al 2012) and AZT (de la Carriére et al 1999;Henderson et al 2012), in accordance with the critical subset model ).…”