Reverse genetics system for studying human rhinovirus infections

Abstract

Human rhinovirus (HRV) contains a 7.2 kb messenger-sense RNA genome which is the template for reproducing progeny viruses after it enters the cytoplasm of a host cell. Reverse genetics refers to the regeneration of progeny viruses from an artificial cDNA copy of the RNA genome of an RNA virus. It has been a powerful molecular genetic tool for studying HRV and other RNA viruses because the artificial DNA stage makes it practical to introduce specific mutations into the viral RNA genome. This chapter uses HRV-16 as the model virus to illustrate the strategy and methods for constructing and cloning the artificial cDNA copy of a full-length HRV genome, identifying the infectious cDNA clone isolates, and selecting the most vigorous cDNA clone isolate to serve as the standard parental clone for future molecular genetic study of the virus.

Figure 1

Strategy for cloning the full-length cDNA of the HRV-16 genome. The genome structure of HRV-16 is shown on the top. The NdeI and BlpI restriction enzyme sites were used for cloning. Viral RNA was extracted from infected cell lysate and converted into cDNA. Three pairs of PCR primers (F1/R1, F2/R2 and F3/R3) were used to generate three overlapping PCR fragments (A, B and C). Each PCR fragment was cloned and the plasmid isolates containing the correct sequences were identified by restriction analysis and sequencing. Then the fragments A, B and C were assembled into a full-length clone with NdeI and BlpI restriction sites.

Figure 2

T7 in vitro transcripts of HRV16 full-length cDNA. T7 in vitro transcripts were made as described in the Method section 3.2.1. Virion RNA was extracted from purified virions as described in the Method section 3.1.3. The concentration of virion RNA was measured with OD_260nm (1 OD = 40 μg/ml). The RNAs were electrophoresed in a 0.8% agarose/1x TBE gel and the gel was stained with EtBr. Lane 1: 1 μg virion RNA, lane 2: 4 μl T7 transcripts, and Lane 3: 2 μl T7 transcripts.