Replication and encapsidation of papillomaviruses in Saccharomyces cerevisiae

Abstract

Improvements in methodologies to recapitulate and study particular biological functions of the papillomavirus life cycle have led to great advances in our knowledge of these viruses. Described in this chapter are techniques that allow low-copy and high-copy replication of full-length human papillomavirus (HPV) genomes, as well as assembly of virus-like particles, in Saccharomyces cerevisiae (yeast). This system has several distinct advantages that make it an attractive complement to the well-established raft-culturing system. First, yeast are inexpensive, rapid, and simple to culture in the lab. Second, they provide an ever-widening array of genetic tools to analyze HPV functions--most recently notable, the yeast open reading frame (ORF)-deletion library. Third, yeast provide a potentially high-efficiency means to produce large quantities of infectious virus in a short time frame. Fourth, assembly of HPV virus in yeast allows encapsidation of mutant genomes, since previous studies have shown that no viral ORF is required for replication of full-length HPV in yeast.

Fig. 1

Human papillomavirus (HPV)/yeast plasmid constructs and HPV ORF expression vectors. Each HPV construct contains a Ura3 nutritional marker placed in a convenient location in the genome. (A) The pPA103 vector, containing the HPV-16 genome, has the Ura3 cassette inserted into nt 7266 of the genome, between the L1 ORF and the LCR. The puc18 backbone can be excised by a BamHI digest. The pPA106 vector, which contains the HPV-31 genome, has the Ura3 cassette inserted into an SpeI site at nt 7559 of the genome, at the 5′ end of the LCR. The pBR322 backbone can be excised by an EcoRI digest. (B) The pPD2-16E2 construct contains a Leu2 selectable marker. The pho5 promoter drives expression of HPV-16 E2 at moderate levels when uninduced. The pL1L2 plasmid contains a bidirectional galactose-inducible promoter, which drives expression of HPV-16 L1 and L2.

Fig. 2

Example of colony formation and episomal replication of HPVs in yeast. (A) Two-hundred ng of plasmid DNA, either a control plasmid (pPA104; puc18-Ura) or pPA103 (HPV16-Ura), was transformed into haploid YPH500 yeast. Yeast were plated on minimal media lacking uracil. Plates were incubated at 30°C for 3 d prior to analysis and quantification of colony formation. (B) Colonies that were capable of growth in the absence of uracil were grown in 5 mL liquid culture overnight, and DNA was isolated. Approx 1 × 10⁸ cell equivalents of DNA was loaded on to a 1% agarose gel and analyzed by Southern blot. Examples of episomal replication of pPA103 (HPV 16) and pPA106 (HPV 31) are shown on the left and right panels, respectively. The controls on the left of each blot represent the number of DNA copies per cell. OC = open-circular, SC = super-coiled.

Fig. 3

Trans-effects of E2 expression on the full-length HPV 16 in yeast; genome amplification and transcription from the viral genome. Yeast containing full-length HPV 16 (pPA103), either expressing HPV 16 E2 (pPD2-16E2) or not (pPD2), were subjected to Southern and Northern analysis to assay for E2-dependent DNA amplification and E6-E7 mRNA production, respectively. pΔYac is included as a negative control in these experiments. Probes for each of the blots are indicated below. The markers to the right of the Northern blot (right panel) represent the nucleotide lengths of RNA markers.

Fig. 4

Virion assembly in yeast. Yeast containing pL1.L2 and pPD2-16E2 or pL1.L2 and pPD2-16E2 and pPA103 (lacking the puc18 vector) were induced to express L1 and L2 open reading frame an d allowed to grow for 1-2 d. Yeast were disrupted by vortex treatment with glass beads, and virus was recovered by differential centrifugation. Virus extracts were analyzed here by electron microscopy using the phospho-tungstenate staining method. Each bar represents 100 nm.