Institute for Molecular Virology1 and Howard Hughes Medical Institute2, University of WisconsinMadison, Madison, Wisconsin 53706, USA
Author for correspondence: Masayuki Ishikawa. Present address: Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. Fax +81 11 706 4932. e-mail ishikawa{at}abs.agr.ac.jp
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The efficiency with which such RNA replicons are transmitted from mother to daughter cells is an important consideration for their possible use for genetic analyses. Moreover, knowing the efficiency of such transfer would provide a foundation for studies of the transmission process, which could shed further light on virus and cell biology. To determine the efficiency with which the B3URA3 RNA replicon was transmitted from 1a2a+ mother to daughter cells, we passaged such yeast in medium containing uracil, thus removing selection for URA3 and the B3URA3 replicon. Specifically, 1a2a+ yeast cells were transfected with B3URA3 in vitro transcripts to obtain Ura+ cells, which were cultured overnight in medium lacking uracil to select for B3URA3. These cells (generation zero) then were inoculated into medium containing uracil. Every 24 h, samples were taken to determine the percentage of Ura+ cells and the cells were subcultured at 1000-fold dilution in fresh, uracil-containing medium. Preliminary studies showed that, when cells reverted to a Ura- (uracil-dependent) growth phenotype under such conditions, such reversion was always associated with loss of the B3URA3 RNA replicon, while retention of the Ura+ growth phenotype was linked to retention of B3URA3. Thus, B3URA3 replicon loss appears to be much more frequent than inactivating mutations of the URA3 ORF or subgenomic mRNA promoter in B3URA3.
As shown in Fig. 2(a) for four independently passaged cultures, plotting log10(Ura+ cells/viable 1a2a+ cells) vs the number of yeast cell divisions yielded straight lines of reproducible slope. These results show that the Ura+ phenotype was lost at a constant rate in each generation, and that the resulting Ura- cells grew at the same rate as the Ura+ cells from which they were derived. From Fig. 2(a)
and multiple independent experiments (e.g. Fig. 3c
,) the frequency of loss of Ura+ phenotype was calculated to range from 5% to 15% per cell division. Thus, at each cell division, the B3URA3 RNA replicon was maintained in 8595% of the progeny of B3URA3-containing cells.
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After the second series of successive passages, we randomly picked 10 Ura+ colonies from each passaged line (total of 40 colonies), amplified them in medium lacking uracil, extracted RNA and analysed it by Northern blot hybridization with a B3URA3 probe. The accumulation level and electrophoretic pattern of B3URA3-related RNAs in these 40 colonies paralleled those in the original B3URA3 RNA-harbouring 1a2a+ cells (Fig. 2c). While these results do not rule out point mutations in the 3a gene, they show that the 3a ORF on B3URA3 RNA was maintained without detectable deletion during continuous replication and mitotic transmission for over 165 yeast generations.
Such long term retention of 3a sequences appeared surprising since the 3a gene is dispensable for RNA3 replication and subgenomic mRNA synthesis (French & Ahlquist, 1987 ; Janda & Ahlquist, 1998
). By contrast, during expression of foreign genes from vectors engineered from many positive-strand RNA viruses, genes not necessary for virus multiplication or spread tend to be quickly deleted, in whole or in part, by recombination (e.g. see Dawson et al., 1989
for tobacco mosaic virus; Mueller & Wimmer, 1998
for polio virus). Similar results have also been found for BMV gene expression vectors (M. Janda & P. Ahlquist, unpublished results). Even for natural viral sequences, a beet necrotic yellow vein virus genomic RNA segment required for fungal transmission but not virus multiplication is quickly shortened by internal deletion when infectious transcripts are mechanically inoculated on a host plant, bypassing selection for fungal transmission (Bouzoubaa et al., 1991
). For BMV, it has been reported that RNA3 derivatives with deletions in the 3a gene accumulate after prolonged incubation of infected barley plants, implying the dispensability and possible negative effect of the 3a gene for the accumulation of BMV in old barley plants (Damayanti et al., 1999
).
Since the 3a gene does not contribute to RNA replication but was maintained in B3URA3 replicons during long term replication and mitotic transmission, these results suggested that 3a function might be necessary for efficient cell-to-cell transmission of BMV RNA replicons from mother to daughter yeast cells. Several independent results appeared consistent with this possibility. In the natural plant hosts of BMV, 3a protein localizes to plasmodesmatal connections between adjacent cells, and is necessary for cell-to-cell movement of infection (Fujita et al., 1998 ; Mise & Ahlquist, 1995
). The ability of 3a protein to cooperatively bind nucleic acids in a sequence-nonspecific manner is thought to be involved in delivering viral RNA from one plant cell to the next (Jansen et al., 1998
; Fujita et al., 1998
), and so might mediate or enhance transmission of B3URA3 RNA from mother to daughter yeast cells.
To test whether the 3a gene contributed to B3URA3 transmission from mother to daughter yeast, we constructed two B3URA3 derivatives with 3a gene disruptions: B3URA3-3afs, with a two-base frameshifting insertion between RNA3 nucleotides 603 and 604, and B3URA3-3a, with a deletion of RNA3 nucleotides 306817 (Fig. 1
; Ahlquist et al., 1981
). As with B3URA3, introduction of either of these derivatives into 1a2a+ yeast generated Ura+ strains that replicated the B3URA3 derivative and readily formed colonies on media lacking uracil. Northern blot analysis (Fig. 3a
, b
) confirmed that these cells contained the expected B3URA3-related RNA replicons and their negative-strand replication intermediates (Janda & Ahlquist, 1993
). When these strains were passaged in the presence of uracil, the frequency of loss of these two 3a-deficient B3URA3 RNA derivatives was similar to or, for B3URA3-3a
, only slightly higher than that of the original B3URA3 RNA (i.e. 1015% per cell division; Fig. 3c
). Thus, the 3a coding region is not required for B3URA3 RNA transmission from mother to daughter yeast and makes little if any contribution to the efficiency of this transmission.
Given the absence of demonstrable selection pressure for the 3a gene in yeast, it is noteworthy that this gene was not deleted from B3URA3 during over 165 cycles of RNA replication and yeast cell division, corresponding to the equivalent of 1045-fold amplification of B3URA3 by RNA-dependent RNA replication. Similarly, though eventually deleted, some unselected foreign gene insertions in positive-strand RNA virus expression vectors have proven more genetically stable than others (Donson et al., 1991 ; Varnavski & Khromykh, 1999
). The detectable appearance of deletion variants in a virus population depends on the frequency of recombination events generating such deletions (e.g. Dawson et al., 1989
; Donson et al., 1991
) and on the replicative fitness of such deletions relative to the starting genome. In other words, even after being generated, a new deletion derivative would remain a rare variant in the population unless it has a replicative advantage over the starting genome. Many foreign gene insertions reduce the replication and/or stability of viral RNAs, giving their deletion derivatives a significant replicative advantage. By contrast, in B3URA3, the wild-type BMV 3a gene may be sufficiently adapted to replication in its RNA3 surroundings that 3a deletions have little or no replicative advantage, as appears to be the case for B3URA3-3a
(Fig. 3a
). Thus, retention of the 3a gene in B3URA3 may be due to the absence of a selective disadvantage, rather than the presence of a selective advantage.
While the BMV 3a gene did not contribute significantly to B3URA3 cell-to-cell transmission in yeast, it remains possible that BMV RNA replicons are actively partitioned to yeast daughter cells by 3a-independent processes. The BMV RNA replication complex is associated with the endoplasmic reticulum (ER), predominantly the perinuclear ER, in both plant cells and in yeast (Restrepo-Hartwig & Ahlquist, 1996 , 1999
). Thus, the ordered segregation of the nuclear envelope (which in yeast remains intact through the cell cycle) and peripheral ER during yeast cell division (Warren & Wickner, 1996
) may transport functional BMV RNA replication complexes and their templates to daughter cells.
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References |
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Received 11 April 2000;
accepted 18 May 2000.
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