L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Abstract

Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus-strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism (9). In this paper, we show that mutations in maklO or the petl8 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.Most strains of the yeast Saccharomyces cerevisiae harbor a 4.5-kilobase double-stranded RNA (dsRNA) called L-A dsRNA. L-A dsRNA is found as intracellular noninfectious viruslike particles (VLPs) whose major coat protein is encoded by L-A dsRNA itself. L-A VLPs are transmitted by cytoplasmic mixing such as occurs during mating (1,12,19).L-A dsRNA requires the products of the chromosomal genes MAK3, MAKIO, and PET18 for its replication or maintenance (18,19,25), and petl8 mutations cause structural instability of L-A dsRNA-containing VLPs (10). These genes also affect maintenance of another dsRNA, called M (1.5 or 1.8 kb, depending on the subspecies), which encodes the killer toxin, a secreted protein lethal to other strains of the same species (3,20,24). A set of six chromosomal genes, called SKI genes, negatively control L-A dsRNA replication (2, 21). L-A dsRNA itself also carries genetic activities ([HOK], [NEX], and [EXL]) in several different combinations, which are defined by their effects on M dsRNA replication (18,19,23,25).Judging by in vivo density transfer experiments, L-A dsRNA replication seems to be conservative (14, 17), similar to that of reoviruses (27) but unlike that of Penicillium stoloniforum virus S, which is semiconservatively replicated (5, 6). In vivo pulse-chase experiments indicate a sequential replication: first plus-strand synthesis, followed by minusstrand synthesis (15).L-A VLPs prepared from stationary-phase cells have one L-A dsRNA molecule per particle and have an RNA polymerase activity whose product is plus-strand L-A singlestranded RNA (ssRNA) which is extruded from the VLPs (4, 7, 11). We call these "mature" or "main-peak" L-A VLPs. Previously, we reported a new type of L-A VLP of lighter density whose in vitro RNA polymerase reaction product was L-A dsRNA (9). These particles were present whe...

The viral killer system in yeast: from molecular biology to application

“…More recently, physical interaction of Mak3p, Mak10p, and Mak31p was demonstrated and it therefore has been proposed that these Mak proteins might also function as a complex in vivo [83,84]. The gene product of PET18 likewise contributes to the overall virion stability, and Pet18p itself is thought to be particle‐associated [85–87]. For stable maintenance of the toxin‐coding M killer virions more than 30 MAK genes have been described which, when mutated, all result in an inability to propagate any toxin‐coding M satellite virus.…”

Molecular organization of Leishmania RNA virus 1.

“…The dsRNA is transcribed into full-length (+)-strands (15,34,35) that are less abundant than the dsRNA and are presumably translated into major coat and coat/RDRP proteins. Particles that contain ssRNA are presumably assembled from the virally encoded proteins and are less dense and have lower sedimentation coefficients than the dsRNA particles (3,35,36). These ssRNA particles synthesize (conservatively replicate) the (-)-strand, making dsRNA (35), thus completing the cycle.…”

Accumulation of viruslike particles in a yeast mutant lacking a mitochondrial pore protein.

“…As an alternative approach, we eliminated VLPs from the original pore mutant by crossing in the maklO mutation, which causes the loss of VLPs from yeast cells (8,23,24 with porin-containing cells (Fig. 7 c and d and Fig.…”