Fig 1.
QRT-PCR strategy to enumerate vRNA and vcRNA species.
Each of the S and L segment viral RNA species generated during LCMV infection are depicted along with the location of the RT primers and PCR primer-probe sets used to quantitate each unique vRNA or vcRNA (for full primer and probe sequences, see Tables 1 and 3). All primer coordinates reflect the relative position on the genomic vRNA. To determine copy number of an individual vRNA or vcRNA species, an RT reaction is performed with a biotinylated primer that has complementarity only to that particular RNA species (e.g. the primer binds the S vRNA, but not the S vcRNA or S-derived subgenomic mRNAs for GPC or NP). The biotinylated cDNAs generated during the RT reaction are affinity purified using magnetic streptavidin beads and subjected to QPCR using the indicated TaqMan primer-probe set. Absolute copy number is determined through the use of a standard curve that is included in each QPCR run. For illustrative purposes, we have depicted the 3’ termini of viral mRNAs as uniformly containing the entire intergenic region. Please note that there is considerable heterogeneity at the 3’ termini of these transcripts as described in [59]. IGR, intergenic region; UTR, untranslated region.
Table 1.
RT primers.
Table 2.
Standard RT-PCR primer sets.
Table 3.
Primers and probe sets for QRT-PCR.
Fig 2.
Validation of TaqMan primer-probe sets.
Each L or S segment-specific TaqMan primer-probe set was tested for its ability to accurately measure known quantities (50, 5x103, 5x105, or 5x107 copies) of a DNA standard control plasmid encoding the target sequence. Depicted are the amplification plots and standard curve fit lines for the primer-probe sets specific for S segment vcRNA (A) or L segment vcRNA (B).
Fig 3.
Nonspecific conversion of arenavirus RNA species into cDNA during RT.
(A) RNA was extracted from i) MC57 cells 7 d after infection with LCMV, ii) cell-free supernatant collected from MC57 cells 18 hr pi with LCMV, or iii) sucrose-banded LCMV particles collected from Vero E6 cells at 48 hr pi. Each sample was subjected to standard RT-PCR targeting the vRNAs or vcRNAs of the L or S segment, as indicated, using the primers described in Tables 1 and 2. In each case, the RT was performed with: i) the RT enzyme and an RT primer, ii) the RT enzyme but no RT primer, or iii) no RT enzyme and no RT primer. It should be noted that the data shown in panel (A) were obtained during the linear and/or plateau phase of the PCR reaction. Therefore, these data are not suitable for exact quantitation, but rather reflect the presence or absence of a particular cDNA. (B). RNA extracted from the samples listed in panel (A) of this figure were subjected to QRT-PCR targeting the vRNAs or vcRNAs of the L or S segment, as indicated, using the primers and probes described in Tables 1 and 3. Similar to panel (A), in each case, the RT was performed with: i) the RT enzyme and an RT primer, ii) the RT enzyme but no RT primer, or iii) no RT enzyme and no RT primer. For each particular viral RNA species, data are presented as mean ± SD relative to the value obtained using the RT primer.
Table 4.
Percent of primer-specific target RNA signal generated in the absence of an RT primer.
Fig 4.
Mapping the nonspecifically-primed cDNA.
RNA extracted from i) Vero E6 cells 48 hr pi with LCMV or ii) sucrose-banded LCMV particles collected from Vero E6 cells at 48 hr pi was subjected to RT in the absence of an RT primer. To determine the extent of nonspecific cDNA conversion of the LCMV S segment, the resulting cDNA products were screened via standard PCR using a panel of PCR primer pairs. Panel (A) shows a depiction of the LCMV S segment vRNA and the region of the segment covered by each PCR primer pair. Primer coordinates reflect the relative position on the S segment vRNA. Panel (B) shows the results of the PCR for each primer pair. Controls included the pT7 S segment plasmid that encodes the intact LCMV S segment as well as a no template control.
Fig 5.
Affinity purification of cDNAs primed with biotinylated RT primers circumvents nonspecific priming during RT.
(A and B) RNA extracted from a high titer stock of cell-free LCMV virions was subjected to standard RT-PCR to detect S segment vRNA using the RT primer S 2865- and PCR primers 1856+ and 2628- (note that the sequence for primer 1856+ is listed in the Methods). In panel (A), three RT conditions were tested. The first RT condition featured a standard RT primer, the second had a biotinylated primer, and the third had no RT primer, as indicated. A portion of each reaction was subjected to affinity purification using streptavidin magnetic beads and then both the input and streptavidin-purified cDNAs were subjected to PCR. In panel (B), two RT conditions were tested: one with a biotinylated RT primer and the other without an RT primer. In an attempt to eliminate nonbiotinylated cDNAs from nonspecifically binding to streptavidin beads, a panel of four wash buffers (the 2X wash buffer from the Dynabeads kilobaseBINDER Kit, a 1X dilution of this buffer alone or containing 0.5% Tween 20, or water containing 0.5% Tween 20) were used during affinity purification. Following affinity purification, the captured cDNAs were subjected to PCR.
Fig 6.
Optimization of assay conditions.
(A) Streptavidin beads do not impact QPCR efficiency. QPCR featuring the primer-probe set specific for LCMV L segment vcRNA listed in Table 3 was conducted using the pT7 plasmid encoding the LCMV L segment. The primer-probe set was tested on a range of plasmid template quantities (50, 5x103, 5x105, or 5x107 copies) with either 25 μl of streptavidin beads (red lines in the amplification plot) or no beads (blue lines in the amplification plot) included in the QPCR reaction mixture. The standard curve fit line was generated by pooling all of the values from samples containing beads or not. Although not shown, similar results were obtained for the remaining primer-probe sets specific for S vRNA and vcRNA or L vRNA. (B) 9 pmol of primer is sufficient to prime total viral RNA template for cDNA synthesis during RT. RNA extracted from sucrose-banded LCMV virions collected from Vero E6 cells at 48 hr pi was subjected to RT using 27, 9, 3, 0.3, or 0 pmol of biotinylated primer S 2865- (to target S vRNA) or L 5906- (to target L vRNA), followed by QPCR using the primer-probe sets for S vRNA or L vRNA, respectively, that are listed in Table 3. Data are presented as mean ± SD relative to the 9 pmol samples. (C) 10 μl of streptavidin beads is sufficient to capture 9 pmol of biotinylated RT primer. RNA extracted from sucrose-banded LCMV particles collected from Vero E6 cells at 48 hr pi was subjected to RT using 9 pmol of biotinylated RT primer S 5906- (specific for L vRNA). Following RT, biotinylated cDNAs were affinity purified using 20, 10, or 5 μl of magnetic streptavidin beads and subjected to QPCR using the primer-probe set for L vRNA listed in Table 3. Data are presented as mean ± SD relative to the 10 μl bead samples.
Fig 7.
Use of optimized QRT-PCR assay to measure the RNA content in LCMV virions and the dynamics of genome replication during acute and persistent LCMV infection.
(A) Viral RNA content of LCMV virions. RNA extracted from sucrose-banded LCMV particles collected from Vero E6 cells at 48 hr pi was subjected to RT using 9 pmol of the biotinylated RT primers listed in Table 1 (gray bars). As a control, RT was also carried out in the absence of an RT primer (black bars). Biotinylated cDNAs were affinity purified using 10 μl of magnetic streptavidin beads and subjected to QPCR using the primer-probe sets for the vRNAs or vcRNAs of the L or S segment that are listed in Table 3. Data are presented as mean ± SD. (B) Dynamics of LCMV replication during acute and persistent LCMV infection. Viral RNA extracted from MC57 cells at 12 h, 18 hr, 7 d, or 24 d pi with LCMV was subjected to RT using 9 pmol of the biotinylated RT primers listed in Table 1. Biotinylated cDNAs were affinity purified using 10 μl of magnetic streptavidin beads and subjected to QPCR using the primer-probe sets for S vRNA or L vRNA, respectively, that are listed in Table 3. Values are listed as mean copies per cell ± SD and are plotted on the left Y axis. For each sample, PFUs in the supernatants were determined by plaque assay and are reported as PFU/cell on the right Y axis.
Table 5.
Ratio of viral RNA species found in virions and infected cells.
Table 6.
Fold-change in LCMV replicative RNA species over the course of infection.