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Current assays for quantification of HIV-1 virions rely on real-time reverse transcriptase (RT)-PCR detection of conserved regions of HIV-1 RNA and can be limited by detection of contaminating viral or plasmid DNA. We developed a novel RT-PCR assay using a reverse primer that hybridizes with the poly(A) tail of HIV-1 mRNAs, anchored by conserved viral nucleotides at the most distal region of the transcript. This assay can detect and quantify HIV-1 RNA with high specificity and sensitivity.
Quantification of virions is important in HIV-1 management and basic research (1–8). Virions are generally quantified using real-time reverse transcriptase (RT)-PCR amplification of genomic RNA with primers in a conserved region, such as gag (9–11). A limitation is lack of specificity because, in principle, any RNA or DNA containing the relevant sequence can be amplified, including integrated or unintegrated viral DNA, and in transfection studies, plasmids containing HIV-1 sequences. Amplification of DNA templates is apparent from positive no-RT controls but nevertheless complicates quantification. In the analysis of HIV-1 RNA in cells, conventional RT-PCR assays can also detect aberrant RNAs, such as chimeric host-virus transcripts originating from upstream host promoters (12, 13) and truncated or prematurely terminated viral transcripts (14–17). Another assay for virions, the enzyme-linked immunosorbent assay (ELISA) detecting HIV-1 Gag p24, has low sensitivity (103 to 106 virus particles/ml) and can also detect virus-like particles lacking genomic RNA and p24 released from dead cells (18–21).
Here we report a new method to quantify virus production and gene expression based on a feature shared by genomic HIV-1 RNA and all spliced HIV-1 RNAs but not aberrant RNAs, integrated or unintegrated proviral DNA, or recombinant plasmids containing HIV-1 sequences. Since all HIV-1 mRNAs, including genomic RNA have a poly(A) tail, we hypothesized that PCR with oligo(dT)-containing downstream primers should allow specific amplification of cDNA generated by reverse transcription of viral mRNA (Fig. 1A). Therefore, HEK293T cells were transfected with the proviral clone pNL4-3, and the viruses produced were collected to extract genomic viral RNA. After reverse transcription using random hexamers as primers, viral RNA was amplified with the primers Tmix and P9285 (see Table S1 in the supplemental material). The amplicon obtained from genomic viral RNA was sequenced to confirm the junction between the terminal portion of the repeat (R) region with the polyadenylation signal and the poly(A) tail. We performed the same amplification and sequencing on plasma virus from 12 HIV-1-infected individuals. A representative experiment showed that viral cDNAs reverse transcribed from genomic viral RNA were readily amplified, but pNL4-3 was not (Fig. 1B). For all 12 patient viruses and NL4-3, the 10 nucleotides immediately upstream of the poly(A) tail were identical (Fig. 1C). For 1,640 sequences from the Los Alamos HIV Sequence Database, the corresponding 10 nucleotides were highly conserved, especially among subtype B viruses (Fig. 1C; see Fig. S1A in the supplemental material). In addition, the forward primer P9501, which spans the region of the TATA box, and the probe, which spans the region of the transactivation response (TAR) element, are also highly conserved (see Fig. S1B and C). Therefore, PCR with a downstream primer consisting of dTs followed by nucleotides complementary to the last few nucleotides before the poly(A) tail should specifically detect all HIV-1 RNAs. We cloned the PCR product from the experiment in Fig. 1B, lane 4, and generated plasmid pVQA, which contains the last 352 nucleotides of viral RNA and 30 deoxyadenosine residues. Using pVQA as the template, we evaluated the efficiency and specificity of oligo(dT)-containing primers. For large copy numbers of pVQA, the efficiency was similar for different oligo(dT)-containing reverse primers from 10T20 to T30 (Fig. 2A). However, with low copy numbers, primers containing 5 to 10 nucleotides complementary to the 3′ end of the R region gave better amplification than a primer containing only 3 complementary nucleotides (3T23) or a primer with 30 dTs. Regarding specificity, 106 copies of pVQA were detected after 20 cycles using each of the oligo(dT)-containing primers, while106 copies of pNL4-3 were detected only with 10T20 or 7T23 and only after 38.5 or 31.4 cycles, respectively (Fig. 2B). Thus, primers with <7 nucleotides complementary to the 3′ end of the R region specifically amplify poly(A)-containing templates rather than proviral DNA. Primer 5T25 provided the best balance between sensitivity and specificity. With 5T25, 10 copies of pVQA could be readily detected, while ≥106 copies of pNL4-3 were required (Fig. 2C). We used a standard (22) containing precisely determined numbers of HIV-1 virions to evaluate the accuracy and sensitivity of this assay. The standard was serially diluted prior to genomic viral RNA extraction and real-time RT-PCR with 5T25. As few as 100 copies of genomic viral RNA could be detected (Fig. 3A). To determine whether this assay could accurately measure viruses in patient samples, especially from the plasma of patients on highly active antiretroviral therapy (HAART), whose plasma HIV-1 RNA levels are <50 copies/ml, we performed a direct comparison between this assay and a single-copy assay (SCA) that has become the definitive assay for residual viremia in patients on HAART whose plasma HIV-1 RNA levels are below the limit of detection of the clinical assay (10, 23). We show that this novel assay can sensitively and accurately quantitate patient viruses both in the supernatants of positive culture wells in viral outgrowth assays and directly in patient plasma (Fig. 3B, ,C,C, and andE).E). However, this assay does require larger input of HIV-1 RNA than the single-copy assay because we failed to detect HIV-1 RNA by this assay when less than 9 ml patient plasma was used (Fig. 3D), which was probably due to the low melting temperature (Tm) value of oligo(dT)-containing primers. The copy numbers of viral RNA obtained with this assay are 1.5- to 3-fold lower than those measured by the single-copy assay, probably because plasmid DNA is used to generate the standard curve, and this does not take into account inefficiency in RNA isolation and reverse transcription. In addition, the presence of terminally truncated genomic RNA with an intact packaging signal may also contribute to the difference in copy numbers measured by these two assays. Therefore, we suggest using an RNA standard for precise measurement (e.g., HIV-1 RNA levels in patient plasma) and using a DNA standard for rapid and relative quantitation (e.g., intracellular viral RNA in the latent reservoir).
To illustrate measurement of virus production with this assay, unstimulated primary CD4+ T cells and primary CD4+ T lymphoblasts obtained by stimulation with anti-CD3 plus anti-CD28 were infected with HIV-1 reporter viruses (24), and culture supernatants were collected for real-time RT-PCR. The HIV-1 burst sizes were 4,680 viruses/cell/day in activated CD4+ T cells and 546 viruses/cell/day in unstimulated CD4+ T cells (Fig. 4A). As a control, we used a budding-deficient virus, NL4-3-Δ6-drGFP. Following infection, viral mRNAs were produced, as evidenced by green fluorescent protein (GFP) expression. However, a premature stop codon in gag prevents virus production. The burst size was <10 viruses/cell/day for both activated and unstimulated cells.
All spliced HIV-1 mRNAs contain a 3′ sequence identical to that of genomic viral RNA and could in principle be detected using oligo(dT)-containing primers (Fig. 4B). Although spliced transcripts are not specifically incorporated into virions due to the lack of a packaging signal (25), it was important to determine whether random incorporation of spliced transcripts affects measurement of genomic viral RNA. Therefore, primers P9501 and 5T25 were used to measure total viral RNA in supernatants from infected Jurkat cells. Spliced transcripts were quantified using gene-specific primers (Fig. 4B; see Table S1 in the supplemental material). As shown in Fig. 4C, vif, vpr, and tat mRNAs represent only 0.5%, 0.4%, and 2.5% of the total virion RNA, respectively. Thus, spliced RNAs do not substantially affect the measurement of virus production using this assay in cell line models of HIV-1 infection.
In conclusion, this viral quantification assay using oligo(dT)-containing primers possesses high specificity and sensitivity for the detection of HIV-1 RNA. It can be applied for measurement of virus levels in patient plasma as well as in laboratory studies of virus production or viral gene transcription.
This work was supported by NIH grant AI043222, the Martin Delaney CARE Collaboratory, the Foundation for AIDS Research (amFAR), the Johns Hopkins Center for AIDS Research, and the Howard Hughes Medical Institute.
Published ahead of print 27 March 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.00006-13.