A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

Abstract

Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules.

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

“…Quantification of ssDNA and dsDNA molecules was performed with a procedure similar to the one described previously for TYLCV and TYLCSV (30). The monomer PepGMV A standard was used to prepare standard curves for the absolute quantification.…”

“…Strandspecific qPCR primers were CS (5=-CATGACGCTGTTGGTGTGGTTCT TG-3=) and VS (5=-CCCATCGTGTAGGCAA GCGTTTCTG-3=). Strandspecific primers were combined with the TAG oligonucleotide (5=-AGTT TAAGAACCCTTCCCGC-3=) for qPCR assays as described previously (30). qPCR efficiency (E) was calculated as follows: E ϭ e (ln10/Ϫs) Ϫ 1, where a slope (s) of Ϫ3.113 represents an efficiency of 109.5% to CS and s of Ϫ3.302 represents an efficiency of 100.8% to VS. Quantification of viral DNA was obtained by extrapolation of C q data with the corresponding standard curve.…”

“…To corroborate the dsDNA nature of the viral DNA present in F1S and F6R samples, we implemented a procedure to quantify viral ssDNA and dsDNA molecules using a qPCR analysis following the protocol described for TYLCV (30). This procedure is based on the independent labeling of both strands of viral DNA (sense and complementary orientations) and its quantification by real-time qPCR.…”

“…This procedure is based on the independent labeling of both strands of viral DNA (sense and complementary orientations) and its quantification by real-time qPCR. According to Rodríguez-Negrete et al (30), in the case of TYLCV, more than 99% of the complementary (Ϫ) strand is present as part of the dsDNA (RF intermediate), whereas the virion sense (ϩ) strand is present mainly in two forms: (i) associated with RF in equimolar concentration with the complementary strand and (ii) as circular, ssDNA free form (and probably most of it encapsidated). Another set of virion sense, ssDNAs could be the molecules identified in two-dimensional (2D) gels for several geminiviruses and proposed to be part of the replicative process (29).…”

“…Production of ssDNA and dsDNA RF is asymmetric during the geminivirus infective cycle. Using a quantitative PCR (qPCR) approach, it has been reported that the dsDNA/ssDNA ratio is about 1:100 in Nicotiana benthamiana and tomato plants infected with tomato yellow leaf curl Sardinia virus (TYLCSV) (30). Therefore, studies on virus minichromosome structure and genome modifications should use methodologies that consider and distinguish the different molecular structures present in the infected cell.…”