Construction of a Transcription Map for Papillomaviruses using RACE, RNase Protection, and Primer Extension Assays

Abstract

Papillomaviruses are a family of small, non-enveloped DNA tumor viruses. Knowing a complete transcription map of each papillomavirus genome can provide guidance for various papillomavirus studies. This unit provides detailed protocols to construct a transcription map of human papillomavirus type 18. The same approach can be easily adapted to other transcription map studies of any other papillomavirus genotype due to the high degree of conservation in genome structure, organization, and gene expression among papillomaviruses. The focused methods are 5'- and 3'-rapid amplification of cDNA ends (RACE), which are techniques commonly used in molecular biology to obtain full-length RNA transcript or to map a transcription start site (TSS) or an RNA polyadenylation (pA) cleavage site. Primer walking RT-PCR is a method for studying the splicing junction of RACE products. In addition, RNase protection assay and primer extension are also introduced as alternative methods in the mapping analysis.

Keywords: RACE; RPA; papillomaviruses; primer extension; primer walking; transcription map.

Figure 1. Identification of HPV18 splice junctions by primer walking RT-PCR

(A) The primers designed for primer walking RT-PCRs to map RNA splicing of HPV18 late transcripts. Arrows below the line diagram for the linearized HPV18 genome are primer positions and orientations. (B) Primer walking RT-PCR products. Poly(A)⁺ total RNA isolated from 16-day-old, HPV18-infected rafts was amplified using three different pairs of HPV18-specific primers (Pr3599 plus Pr5793, Pr5939, or Pr5628). Products 1 to 4 were gel purified, cloned, and sequenced. Shown below the gel are the corresponding splice junctions identified by sequencing. The products 2 and 3 in the gel are major late transcripts (L1) spliced from nt 3696 to 5613. This splice junction could be also verified by using a backward splice junction primer, Pr5628, of which the 3′ end has 2 nt identical to nt 3696 to 3695, giving the product 4 in the gel. The product 1 in the gel, detected with a primer pair of Pr3599 and Pr5793, is a product spliced from nt 3786 to 5776. This minor transcript is detectable only with a primer pair of Pr3599 and Pr5793 because the 3′-end 3 nt of Pr5793 are identical to the sequences from nt 3786 to 3784 and thereby the Pr5793 primer can function as a splice junction 3786/5776 primer. This figure is modified with permission from a reference (Wang, et al., 2011).