Family: Pseudoviridae
Genus: Pseudovirus
Distingushing features
The genus Pseudovirus is currently established based upon the length of the tail of the tRNA molecule that is used as a primer to initiate reverse transcription. However, classification only based on this feature is inconsistent with phylogenetic analyses, which reveal the current members of this genus to be polyphyletic. The demarcation criteria for the genus are therefore likely to change in the future.
Virion
Morphology
Virions of Saccharomyces cerevisiae Ty1 virus (SceTy1V) are ovoid to spherical, strictly intracellular particles, which are sometimes observed to organize into paracrystalline clusters in the cytoplasm of yeast cells. Cryo-electron microscopy analysis of the SceTy1V virus-like particles (VLPs) reveals that they have an icosahedral symmetry (Figure 1. Pseudoviridae). Particles of SceTy1V are structurally heterodispersed. However, a C-terminal deletion mutant containing residues 1–381 of the Gag polyprotein is less heterodisperse than the wild-type particles. The mean radius of the virions is 20–30 nm for wild type and 20 nm for the much better characterized and more uniform 1–381 mutant. Virions are not enveloped.
Physicochemical and physical properties
The virion Mr is estimated to be 14 MDa with a sedimentation coefficient of 200–300S for the wild type and 115S for the 1–381 mutant. Virions are sensitive to high temperature (65°C). The major virion RNA species of SceTy1V consists of a long terminal repeat (LTR)-to-LTR transcript of 5.6 kb. In addition, SceTy1V packages host-derived primer tRNAMet. VLP preparations contain a mixture of RNA and DNA molecules, where the RNA represents the major nucleic acid form.
Genome organization and replication
The RNA genome of SceTy1V is 5.6 kb, or 5.9 kbp as integrated DNA. The known encoded proteins in the gag gene are CA (capsid) and possibly a short C-terminal peptide which has not been directly observed. No recognizable nucleocapsid (NC) peptide has been identified, although the C-terminal Gag peptide may perform a similar function. The proteins encoded by the pol gene are protease (PR), integrase (INT) and reverse transcriptase-ribonuclease H (RT-RH). All of these proteins are required for replication. There is evidence that cleavage at the Gag-PR boundary obligatorily precedes the other cleavages. The DNA form of the SceTy1V genome consists of two 335 nt LTRs flanking the central coding region. The LTR sequences can be divided into three segments called U3, R and U5 (Figure 2. Pseudoviridae). The Gag-Pol precursor is expressed by an inefficient programmed frameshift that occurs within the sequence 5′-CUU AGG C-3′ (the indicated codon boundaries represent the Gag frame), which is necessary and sufficient to specify the +1 frameshifting. A minor shorter transcript of about 2.2 kb is reported to be 5′-co-terminal with the major transcript but has not been fully characterized. A second minor transcript is reported to be 3′-co-terminal with the major transcript and is most clearly observed in yeast strains with spt3 mutations. These spt3 mutations eliminate or greatly reduce the abundance of the full-length transcript. The initial step in replication is transcription of the SceTy1V genome to generate a full-length RNA that is encapsidated in the cytoplasm into a precursor particle consisting of unprocessed Gag and Gag-Pol proteins. Action of the SceTy1V PR then converts it into a mature virus particle. It is thought that this activates the reverse transcription process.
The first step in the reverse transcription process is the extension of the initiator tRNAMet primer, which binds to the (−) strand primer binding site ((−)PBS) in the full-length RNA. The product of this extension is referred to as (−) strand strong stop DNA or (−)ssDNA, in analogy with retroviruses. The (−)ssDNA is transferred to the 3′-end of the full-length RNA, where it can be further extended to generate a nearly full-length (−) strand DNA. Priming of the plus strand initiates at the (+) PPT1 (for polypurine tract 1) adjacent to the 3′-LTR, but the mechanism of this priming and the exact nature of the primer have not been determined. Extension yields a product, (+)ssDNA that corresponds to the similarly named retroviral intermediate. Transfer of (+)ssDNA to the left end of the (−) strand DNA sets up a primer-template that can be extended, in principle, to generate the full-length duplex DNA. However, studies indicate that the (+) strand is not continuous because a second priming event occurring near the middle of the molecule at a site called (+) PPT2 results in a discontinuity in the middle of the (+) strand. We refer to this final product of reverse transcription as the dsDNA form, although some experiments suggest that in many of these molecules there may be stretches of RNA rather than DNA in the (+) strand. The dsDNA is imported into the cell nucleus, possibly by a nuclear localization signal found at the C-terminus of INT. The full-length dsDNA is a substrate for the SceTy1V INT, which inserts the dsDNA into a chromosomal target site, in the process generating a 5 nt duplication of the host target site DNA. Sequences located upstream of the RNA polymerase III-transcribed genes represent strongly preferred targets for such SceTy1V integration in vivo.
Biology
SceTy1V can be thought of as a genome parasite of Saccharomyces cerevisiae. In typical wild strains of this yeast, 3–15 copies of SceTy1V are found per haploid genome, whereas in typical laboratory isolates, there are 25–40 copies. In addition to these complete copies, the genome contains several hundreds of solo-LTRs (LTRs not associated with a central coding region) or fragments thereof. SceTy1V appears to be restricted to S. cerevisiae and to very closely related species but is absent from more distantly related members of the genus. However, those other species are likely to harbour related viruses. Transmission is likely to be exclusively vertical. SceTy1V and members of closely related species, such as the virus Saccharomyces cerevisiae virus Ty2 (SceTy2V), have been both found in virtually all isolates of S. cerevisiae, from all over the world. No cytopathic effects have been reported. Some strains contain large numbers of SceTy1V virus particles and are otherwise normal in every way. Overexpression of SceTy1V proteins leads to slow growth, but this phenotype is poorly characterized.
Species demarcation criteria
See discussion under family description.