Yeast Virus Propagation Depends Critically on Free 60S Ribosomal Subunit Concentration

Abstract

Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M 1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M 1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3 poly(A).Studies of viral propagation generally concentrate on the activities and interactions of virus-encoded proteins. However, the host environment and particular host components can play a crucial role in whether viral propagation is persistent or causes pathology to the host. This is amply demonstrated by studies of the roles of cellular components in bacteriophage replication and assembly.Studies of the L-A double-stranded RNA (dsRNA) virus and its killer toxin-encoding satellite, M 1 dsRNA, have identified many genes of its host, Saccharomyces cerevisiae, which either repress viral propagation (the SKI genes, so named for the superkiller phenotype of mutants in these genes) or are necessary for M 1 propagation (MAK genes, for maintenance of killer). Recently, the SKI2, SKI3, and SKI8 genes have been shown to act by specifically repressing the translation of nonpoly(A) mRNAs, such as the L-A and M 1 mRNAs (28a). In this report, we examine the mechanism by which most of the MAK genes promote viral propagation.The L-A dsRNA virus of S. cerevisiae has two open reading frames in its positive strand, the 5Ј gag encoding its major coat protein (Gag) and the 3Ј pol encoding the multifunctional Pol domain of the Gag-Pol fusion protein. M 1 is a dsRNA satellite of L-A, depending on the L-A-encoded Gag and Gag-Pol for its propagation. M 1 itself encodes no proteins required for its propagation but does encode the killer toxin, a secreted heterodimer protein processed out of a 32-kDa preprotoxin. The L-A genome lacks both 5Ј cap and 3Ј poly(A) (10, 42), and viral transcripts made in vitro lack both modifications, although a single uncoded A (or G) residue is found at the 3Ј end of both viral strands (11,42).Mutants isolated by their inability to propagate the M 1 dsRNA identify about 30 chromosomal MAK genes. MAK3 encodes an N-acetyltran...

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

“…Deletion of either RPL8A or RPL8B had a detectable growth defect ( Fig. 1A; Yon et al 1991;Ohtake and Wickner 1995). Previously, Robledo et al (2008) showed that knockdown of human L7 led to a decrease in 60S subunits.…”

“…As is the case for many r-proteins (Mager et al 1997), both L7 and L8 are encoded by two different, unlinked genes (Arevalo and Warner 1990;Yon et al 1991;Ohtake and Wickner 1995). Therefore, we began by deleting each copy of the RPL7 and RPL8 genes to determine which deletion of each pair had the stronger phenotype.…”

Ribosome Concentration Contributes to Discrimination against Poly(A)− mRNA during Translation Initiation in Saccharomyces cerevisiae

“…Similarly, some, but not all, mutations that reduce ribosomal subunit levels reduce the expression of poly(A) Ϫ yeast viral mRNAs (44). In both cases, 60 S subunit changes produced a more pronounced effect, possibly reflecting a more critical role of the 60 S subunit joining step in enhancing translation of poly(A) ϩ mRNAs.…”

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

“…The killer phenotype is caused by infection of yeast cells by the endogenous L-A and M 1 dsRNA viruses (reviewed in Wickner 1996). The M 1 virus, and hence the killer phenotype, is highly susceptible to defects in the translational apparatus, e.g., to defects in 60S ribosomal subunit biogenesis (Ohtake and Wickner 1995b), to the presence of translational inhibitors (Sommer and Wickner 1982;Carroll and Wickner 1995), to mutations in ribosomal proteins Carroll and Wickner 1995;Ohtake and Wickner 1995a;Meskauskas and Dinman 2001), and to changes in the efficiency of programmed −1 ribosomal frameshifting (−1 PRF; Dinman and Wickner 1992). In order to minimize recombination and select cells containing only mutant forms of 5S rRNA, a rigorous selection scheme involving negative selection for both the wild-type RDN1 gene and the plasmid on which it was encoded was devised (see Materials and methods).…”