Asynchronous Accumulation of Lettuce Infectious ... - Journal of Virology

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and B1 show longer exposures for the respective lanes, and the genomic RNAs (G) are indicated. Images were arranged and labeled using Adobe Photoshop ...
JOURNAL OF VIROLOGY, July 2000, p. 5762–5768 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 13

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation HSIN-HUNG YEH, TONGYAN TIAN, LUIS RUBIO, BRETT CRAWFORD,

AND

BRYCE W. FALK*

Department of Plant Pathology, University of California, Davis, California 95616 Received 28 January 2000/Accepted 17 April 2000

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation. Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. The family Closteroviridae contains two genera: the genus Closterovirus and the genus Crinivirus. Viruses in the genus Closterovirus are generally transmitted by aphids and have large (15,000 to 20,000 nucleotides) single-stranded RNA monopartite genomes, while viruses in the genus Crinivirus are transmitted by whiteflies and have bipartite genomes of ca. 15,000 nucleotides (14). LIYV is the only member of the genus Crinivirus for which the complete genome nucleotide sequence is available (12). Viruses in the family Closteroviridae have several distinct characteristics relative to those of plant viruses in other taxonomic groups. Infections are mostly limited to phloem tissues of their host plants, and the viruses are absolutely dependent on their phloem-feeding insect vectors for plant-to-plant transmission. The viral genomes are the largest of the singlestranded plus-sense RNA plant viruses, and they contain a large number of genes (10 to 12), many of which are unique to and conserved among the viruses in this family (1; see Fig. 1). All contain genes encoding a papain-like leader protease, domains which are common for Sindbis-like virus replicationassociated proteins (methyltransferase, helicase, and RNA-dependent RNA polymerase), and the “closterovirus hallmark gene array” (6). This gene array includes the following: a gene encoding a small hydrophobic protein; the gene encoding the heat shock protein 70 homolog (HSP70), a protein of ca. 60 kDa (p59 for LIYV); and genes encoding the capsid protein (CP) and the minor capsid protein (CPm). Finally, closterovi-

rus genomic RNAs are encapsidated in morphologically polar capsids composed of the CP and CPm (2, 7, 23). Although the biological functions of proteins encoded by the conserved closterovirus hallmark gene array have yet to be proven unequivocally, evidence suggests that they are not involved in genomic RNA replication. The Beet yellows virus (BYV; genus Closterovirus) HSP70 homolog is likely involved in inter- and intracellular trafficking (3, 15, 18), and the BYV and LIYV HSP70 homologs are virion associated (15, 23). Recent immunoneutralization experiments suggest that the CPm may be a primary determinant involved in transmission of LIYV by the whitefly, Bemisia tabaci (23). The LIYV closterovirus hallmark gene array is contained in RNA 2, and comparative sequence analyses suggested that the LIYV replicationassociated proteins were encoded by LIYV RNA 1 (12). Subsequent experiments showed that LIYV RNA 1 alone was replication competent while RNA 2 was replicated only when it was coinoculated with RNA 1 (11). Likewise, cDNA constructs for BYV and Citrus tristeza virus (CTV; genus Closterovirus) in which the closterovirus hallmark gene array region has been deleted yielded transcripts which were replication competent (17, 21). For BYV, the only open reading frames (ORFs) contained by the minimal replication-competent constructs were ORFs 1A and 1B. Still, some evidence suggests that in addition to the ORFs 1A- and 1B-encoded proteins, other virus-encoded proteins may be involved in the replication of these large genomic RNAs. For the Closterovirus BYV P21 encoded by the most 3⬘-terminal ORF was identified as a replication enhancer (17). Interestingly, all viruses in the family Closteroviridae studied so far encode at their 3⬘ termini a protein similar in size to BYV P21. However, only BYV P21 and CTV P23 have been reported to have sequence similarity (17). LIYV has several distinct features which suggest that its

* Corresponding author. Mailing address: Department of Plant Pathology, 1 Shields Ave., University of California, Davis, CA 95616. Phone: (530) 752-0302. Fax: (530) 752-5674. E-mail: bwfalk@ucdavis .edu. 5762

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replication strategies may differ not only from viruses in the genus Closterovirus but also from most other multipartite RNA viruses. First, the bipartite nature of the LIYV genomic RNAs contrasts with the monopartite genomic RNAs for viruses in the genus Closterovirus. If the LIYV replication-associated proteins are encoded exclusively by RNA 1, then RNA 1 is likely replicated in cis while RNA 2 is replicated in trans. Second, there is surprisingly little sequence homology between LIYV genomic RNAs 1 and 2. The 5⬘-terminal five nucleotides of each genomic RNA are identical, and a 23-nucleotide sequence near the 5⬘ terminus is shared. However, the 3⬘-terminal sequences of the two LIYV genomic RNAs are distinct, and computer analysis suggests that there is no similar secondary structure between LIYV RNA 1 and RNA 2 3⬘ termini (12). These features raised questions about the replication of LIYV RNA 2. Does the same LIYV replication complex recognize RNA 1 and RNA 2 equally? Does LIYV RNA 2 replication and accumulation require proteins other than those encoded by RNA 1 ORFs 1A and 1B? In this paper, we show an asynchronous accumulation pattern for LIYV genomic RNAs 1 and 2 when they are inoculated simultaneously to protoplasts. Mutagenesis studies also showed that RNA 2-encoded proteins do not affect RNA 1 and/or RNA 2 accumulation. However, mutations in the LIYV RNA 1 3⬘-terminal ORF (ORF 2 encoding P32) did not affect RNA 1 accumulation, but severely reduced accumulation of LIYV RNA 2. MATERIALS AND METHODS Construction of LIYV mutants. Most mutants were constructed by using sitedirected mutagenesis to insert two in-frame stop codons near the 5⬘ termini of specific LIYV ORFs by using the transformer site-directed mutagenesis kit (Clontech) according to manufacturer’s recommendations. Full-length cDNA clones to LIYV RNA 1 (pSP9/55) and RNA 2 (pSP6) (11) were used for mutagenesis when possible. However, mutagenesis on the CP and CPm ORFs was done by first excising a 941-bp fragment (nucleotides 4407 to 5348) from pSP6. This fragment was subcloned into SalI-HindIII-digested pBluescript SK II (Stratagene), and mutagenesis was performed using primers shown in Table 1. Mutated fragments were then reinserted into pSP6. Double mutants for the CP and CPm ORFs (CPPM⫺) and the HSP70 and p59 ORFs (HP⫺) were also constructed by ligating fragments together which contained each individual mutation. The RNA 1 Rep⫺, P32 F1, and P32 F2 mutants were constructed by first excising and discarding the EcoRI fragment between nucleotides 448 and 5180 from pSP9/55 (pEco RI⫺). For the Rep⫺ mutant, site-directed mutagenesis was then used to modify the overlapping region of ORF 1B and ORF 2 (encoding P32) in pEco RI⫺. The P32 F1 and P32 F2 frameshift mutants were generated by digesting pEco RI⫺ with XbaI and NdeI, respectively. The digested plasmids were then treated with Klenow and religated. This served to introduce 4 and 2 nucleotides, respectively, into ORF 2 of P32 F1 and P32 F2, resulting in frameshifts and stop codon insertions within the P32 coding region (Table 1). The LIYV RNA 1 EcoRI fragment (nucleotides 448 to 5180) was then reintroduced into the modified plasmids to yield the Rep⫺, P32 F1, and P32 F2 mutants. The final mutant, RNA 1 P32⫺, was constructed by PCR amplification of a 911-bp fragment (nucleotides 7208 to 8118) corresponding to ORF 2. The forward primer (Table 1) was designed to insert two in-frame stop codons into ORF 2. The PCR product was digested with MfeI and NotI and ligated into MfeI-NotIdigested pSP9/55 to yield the RNA 1 P32⫺ mutant. Thus, these approaches resulted in each mutant either containing a new or lacking a wild-type restriction enzyme site so that specific mutants could be monitored (Table 1). All mutant clones were sequenced in the modified region in order to ensure that the mutation was correct. Specific constructions for all mutations are shown in Table 1, and their relative positions are indicated in Fig. 1. LIYV inocula and protoplast manipulation. LIYV virions were purified from LIYV-infected Chenopodium murale plants, and RNAs were extracted as previously described (10). Capped transcripts corresponding to wild-type and mutant LIYV RNAs 1 and 2 were synthesized as previously described (11). Protoplasts were prepared from cultured Nicotiana tabacum suspension cells (16) and inoculated using 5 ␮g of each transcript essentially as previously described (13), except that 1.2 million cells were used for each inoculation, and after inoculation, protoplasts were incubated at 26.5°C. Analysis of wild-type and mutant LIYV replication. LIYV-inoculated protoplasts were collected and analyzed by methods similar to those previously described (11). Aliquots containing ca. 1.2 ⫻ 105 cells were collected by centrifugation (1,310 ⫻ g) at different times postinoculation (p.i.), and RNAs were

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isolated using TRI Reagent (MRO) according to the manufacturer’s recommendations. Generally, RNAs representing ca. 1.2 ⫻ 104 cells were used for Northern hybridization analysis. RNAs were denatured with glyoxal, separated by agarose gel electrophoresis, and transferred to Hybond NX (Amersham) as previously described (10). DNA fragments corresponding to nucleotides 7589 to 8118 of LIYV RNA 1 and nucleotides 6685 to 7193 of RNA 2 were excised from pSP9/55 and pSP6 by using XbaI and NotI and ligated into SpeI-NotI-digested pBluescript II SK(⫹) (Stratagene), yielding pSKL1 and pSKL16, respectively. T3 RNA polymerase and EcoRI-digested plasmids were used to generate negative-sense digoxigenin (DIG)-labeled probes, and T7 RNA polymerase and NotI-digested plasmids were used to generate positive-sense DIG-labeled probes (Boehringer Mannheim) for RNAs 1 and 2 (from pSKL1 and pSKL16, respectively). Immobilized LIYV RNAs were subjected to hybridization (11), and positive hybridization reactions were detected by using the chemiluminescent substrate CDP STAR (Boehringer Mannheim) and by exposing blots to Fuji medical X-ray film. Hybridization signals were quantified by scanning the exposed film to calculate the optical density using an IS-1000 digital imaging system (Alpha Innotech Corp.). In order to ensure that optical density signals were in the linear range, a dilution series of LIYV RNA 1 in vitro transcripts ranging from 0.35 to 25 ng were included as internal standards.

RESULTS Temporal accumulation of LIYV RNAs 1 and 2. In order to monitor LIYV RNA accumulation, we first assessed the time course accumulation of LIYV RNAs in LIYV-inoculated protoplasts. When protoplasts inoculated with LIYV virion RNAs (containing both genomic RNAs 1 and 2) were analyzed, differential temporal accumulation of LIYV RNAs 1 and 2 was observed. Full-length negative-sense RNA 1 (Fig. 2C, lane 12) and the subgenomic RNA corresponding to the RNA 1 ORF 2 (encoding P32; Fig. 2A, lane 12) were detected at 12 h p.i. Longer exposures also showed accumulation of the positivesense genomic LIYV RNA 1 at 12 h p.i. (Fig. 2A); however, it was much more abundant by 24 h p.i. Time course comparisons showed that the P32 subgenomic RNA and negative-sense genomic RNA 1 reached maximum accumulation at 24 h p.i., but positive-sense genomic RNA 1 continued to accumulate up to the final sampling time of 72 h p.i. (Fig. 2A and C). Optical density analysis of exposed films showed that the positive-sense LIYV genomic RNA 1 increased ca. 11-fold between 12 and 24 h p.i., but then only a twofold increase was seen between 24 and 72 h p.i. In contrast, LIYV RNA 2 temporal accumulation was much different from that seen for RNA 1. Positive- and negativesense full-length LIYV RNA 2 did not accumulate as early p.i. as did RNA 1. RNA 2 input inoculum was detected at 0 h p.i.; however, the amount of RNA 2 detected at 12 h and even 24 h p.i. was less than that at 0 h p.i. (Fig. 2B). The amount of LIYV RNA 2 detected at 24 h p.i. always appeared slightly greater than at 12 h p.i. (less than twofold; Fig. 2B). This is in contrast to what was seen for RNA 1; both positive- and negative-sense full-length RNA 1 showed large increases by 12 h p.i., much greater than the amount of input inoculum (Fig. 2A and C). Accumulation of both positive- and negative-sense LIYV RNA 2 increased rapidly (ca. 16-fold) between 24 and 36 h p.i. and continued to increase, albeit at a lower rate, until the end of the sampling (ca. twofold between 36 and 72 h p.i.; Fig. 2B). Interestingly, the pattern of accumulation for a LIYV RNA 2 defective RNA (20) was similar to that of genomic RNA 2 (Fig. 2B and D). When these experiments were repeated using in vitro-derived LIYV RNA 1 and 2 transcripts as inocula, the patterns of accumulation were indistinguishable from those seen here for virion RNAs (not shown). Mutational analysis of LIYV RNA 2. Although LIYV RNA 1 can replicate in protoplasts and accumulate to high levels in the absence of RNA 2 (11; Fig. 3A), the role(s) of RNA 2 and/or its encoded proteins in the replication and accumulation of LIYV RNAs is not known. Therefore, we constructed

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J. VIROL. TABLE 1. LIYV mutant clones and oligonucleotides used for mutagenesis

Clonea

Primer and modified sequenceb

Affected ORF

Rep⫺

Three-amino-acid change in ORF 1B and truncation in P32

A A L C Y I H C C C GCA GCA TTG TGC TAT ATA CAC TGT TGC A A L S L R H C C C GCA GCA TTG AGC TTA AGA CAC TGT TGC 29 AflII

P32⫺

Truncation in P32

57 TT ATG ACA ATT GTG AAA TTG TGT GGA TTT AAG ATG GAC ACT TCA AGT TTT ATC GC TT ATG ACA ATT GTG AAA TTG TGT GGA TTT AAG ATG TAA ACT TAA GGT TTT ATC GC AflII

P32 F1

Frameshift and truncation of P32

XbaI TTC TCT AGA TAC TAT GTC TTC TCT AGC TAG ATA CTA 174

P32 F2

Frameshift and truncation of P32

NdeI GAA TAC AAT GAC ATA TGT ACG GTC TAC AAT TTG AAA GAA TAC AAT TAC ATA TAT GTA CGG TCT ACA ATT TGA 237

HSP⫺

Truncation in HSP70 homolog

35 AT TCG GCT TAC ATA CCA ACG TGT ATT GCT AT TCG GCT TAA GTA CCA ACG TGA ATT GCT AflII

P59⫺

Truncation in P59

70 GTC TCT CCC AAA GAC AGT AAT GCC TAT GTG AAA C GTC TCT CCT TAA GAC AGT AAT GCC TAA GTG AAA C Af1II

CP⫺

Truncation in CP

91 C AAA GAC CCC AAC AGA AAT CAA CCT AGT G C AAA GAC CCC TAG TGA TAT CAA CCT AGT G EcoRV

CPm⫺

Truncation in CPm

71 XbaI TCA ACA TCT ACA TCT AGA AGT ATT TTT CAA C TCA ACA TCT ACA TAG TGA AGT ATT TTT CAA C

P26⫺

Truncation in P26

39 C ATC TCT TCT AAA CAT GGG AAA AGT GTC GC C ATC TCT TCT TAA GAT GGG TAA AGT GTC GC AflII

a Name of corresponding mutant. Rep⫺ has mutations in the overlapping ORF 1B (three amino acid changes indicated in bold) and ORF 2 (two stop codons indicated by underlines). Positions of mutations within the genomic RNAs are also indicated in Fig. 1. b The upper nucleotide sequence represents the wild-type LIYV sequence, and the lower nucleotide sequence shows the oligonucleotide used for mutagenesis and resulting nucleotide sequence. For the P32 F1 and P32 F2 frameshift mutants, the lower sequence represents the resulting sequence and bold italic letters are those inserted by the mutagenesis strategy. Triplets represent codons within the ORF, and the number indicates the position of the amino acid residue encoded by that codon within the ORF. Bold letters indicate the restriction enzyme site introduced or eliminated by the mutagenesis. Underlined letters indicate the resulting stop codons. Encoded amino acids are indicated for the Rep⫺ mutant, and the wild-type (upper) and mutant (lower) amino acid sequences are shown (single-letter code). As the RNA 1 ORF 1B and ORF 2 overlap in separate reading frames, the Rep⫺ mutation affects both ORFs. Triplets shown are for ORF 1B, but the indicated stop codons (underlined nucleotides) affect ORF 2, and thus, underlined nucleotides and corresponding amino acid residue number, 29, are for ORF 2.

mutations in specific RNA 2 ORFs and assessed effects of these mutations on LIYV RNA accumulation in protoplasts. Protoplasts were inoculated with wild-type RNA 1 transcripts alone, wild-type RNA 1 and RNA 2 transcripts, or wild-type LIYV RNA 1 transcripts plus transcripts of specific LIYV RNA 2 mutants. The relative accumulation of LIYV genomic RNAs 1 and 2 and three subgenomic RNAs was assessed for wild-type and mutant constructs at 24 and 48 h p.i. (Fig. 3). RNA hybridization analyses showed that the accumulation of LIYV RNA 1 and RNA 2 genomic and subgenomic RNAs varied slightly from experiment to experiment. However, based on four replicated experiments, accumulation of the LIYV RNA 1 genomic and ORF 2 (encoding P32) subgenomic RNA

and the RNA 2 genomic and subgenomic RNAs for the HSP70 homolog and P26 ORFs were not different whether the inocula contained wild-type or LIYV RNA 2 mutant RNAs (single or double mutants; Fig. 3A and B, respectively). Thus, these data suggest that the RNA 2 ORFs encoding the HSP70 homolog, P59, CP, CPm, and P26 did not affect accumulation of the LIYV genomic RNAs 1 or 2 or the P32, P26, or HSP70 homolog subgenomic RNAs. Mutations in LIYV RNA 1 ORF 2 affect accumulation of RNA 2 but not RNA 1. Computer-assisted sequence analyses suggested that LIYV RNA 1 ORFs 1A and 1B encode the proteins associated with LIYV RNA replication (12), and infectivity studies shown here (Fig. 3A) and previously (11)

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FIG. 1. Schematic representation of the LIYV genomic RNAs and specific LIYV mutants. Rectangles represent ORFs in LIYV genomic RNAs 1 and 2. LIYV RNA 1 ORFs 1A, 1B, and 2 are shown. P-PRO, papain-like protease; MTR, methyltransferase; HEL, RNA helicase; RDRP, RNA-dependent RNA polymerase; HSP70, homolog of HSP70 proteins. Arrows indicate positions in LIYV genomic RNAs where mutations were introduced (Table 1).

clearly demonstrate that RNA 1 alone is replication competent. However, in addition to ORFs 1A and 1B, LIYV RNA 1 encodes in ORF 2 P32 a protein of unknown function and for which no significantly similar proteins have been identified in database searches (data not shown). In order to assess the

potential role(s) of ORF 2 and/or P32 in LIYV replication, we created four mutations in LIYV RNA 1 ORF 2 and compared accumulation of LIYV RNAs 1 and 2 in protoplasts. The first mutant (RNA 1 Rep⫺; Table 1) was constructed to yield three amino acid changes in the ORF 1B-encoded protein and to

FIG. 2. Time course of accumulation of LIYV RNAs in protoplasts. LIYV virion RNAs were used to inoculate 1.2 ⫻ 106 tobacco protoplasts, and 1.2 ⫻ 105 protoplasts were collected at the times p.i. indicated above respective lanes. Lane 0, sample taken immediately after inoculation. Total RNAs from 1.2 ⫻ 104 protoplasts were used for Northern blot hybridization with a DIG-labeled negative-sense RNA 1 probe (A), positive-sense RNA 1 probe (C), negative-sense RNA 2 probe (B), and positive-sense RNA 2 probe (D). RNAs corresponding to the LIYV genomic RNAs are indicated by G, the RNA 1 P32 subgenomic RNA is indicated by SG, and the RNA 2 defective RNA is indicated by D. Numbers at the left correspond to positions of marker RNAs (sizes in nucleotides) analyzed in the same gel. Inserts A1 and B1 show longer exposures for the respective lanes, and the genomic RNAs (G) are indicated. Images were arranged and labeled using Adobe Photoshop 4.0.

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FIG. 3. Accumulation of wild-type and LIYV RNA 2 mutant RNAs in protoplasts. Transcripts of wild-type LIYV RNA 1 (from clone pSP9/55) were inoculated alone, with wild-type LIYV RNA 2 (pR6) transcripts, or with specific LIYV RNA 2 mutant transcripts (Fig. 1 and Table 1) to 1.2 ⫻ 106 tobacco protoplasts, and 1.2 ⫻ 5 10 protoplasts were collected at 24 and 48 h p.i. Total RNAs purified from 1.2 ⫻ 104 protoplasts collected at 24 and 48 h p.i. were used for Northern blot hybridization with RNA 1 (A) and RNA 2 (B) negative-sense DIG-labeled probes. Labels above lanes indicate the inocula used for the given sample. Lanes: RNA1, only RNA 1 transcripts; WT, wild-type LIYV RNA 1 (from pSP9/55) and RNA 2 (from pSP6) transcripts. Remaining labels indicate wild-type RNA 1 plus specific LIYV RNA 2 mutants (see Table 1 for mutants; HP⫺ is a double mutant for both the HSP70 homolog and p59 ORFs, and CPPm⫺ is a double mutant for both the CP and CPm ORFs). Numbers at left correspond to positions of marker RNAs (sizes in nucleotides) analyzed in the same gel. Arrows labeled P32, HSP70, and P26 indicate hybridization signals for the corresponding subgenomic RNAs. Images were arranged and labeled using Adobe Photoshop 4.0.

introduce two stop codons into the 5⬘-terminal region of ORF 2 (residues 29 and 31), as ORFs 1B and 2 overlap (Fig. 1). When transcripts of RNA 1 Rep⫺ were inoculated alone (not shown) or with wild-type RNA 2 transcripts to protoplasts, no LIYV RNA accumulation was detected (Fig. 4), suggesting that this mutation was lethal. The second mutation (LIYV RNA 1 P32⫺) was localized to only RNA 1 ORF 2 and truncated this ORF by inserting two stop codons for residues 57 and 59 (Fig. 1). RNA hybridization analysis of inoculated protoplasts showed that LIYV RNA 1 P32⫺ accumulated to levels indistinguishable from those of wild-type LIYV RNA 1, and similar levels of the P32 subgenomic RNA were also detected (Fig. 4A). However, coinoculation of LIYV RNA 1 P32⫺ plus RNA 2 transcripts resulted in very low levels of LIYV RNA 2 accumulation (Fig. 4B). In some experiments, no RNA 2 accumulation was detected, while in most, RNA 2 accumulated to only 3 to 10% of the level seen for RNA 2 coinoculated with wild-type RNA 1. Further proof that this was newly replicated LIYV RNA 2 was obtained by hybridization analyses for negative-sense LIYV RNA 2. Low levels of negative-sense LIYV RNA 2 progeny were detected when the inoculum contained wild-type RNA 2 and LIYV RNA 1 P32⫺. As this inoculum contained only positive-sense LIYV RNA 2 transcripts, the negative-sense products could only result from RNA 2 replication (not shown). Also, comparison of LIYV RNA 2 detection in protoplasts coinoculated with the LIYV RNA 1 P32⫺ and the RNA 1 Rep⫺ mutants showed that low levels of positive-sense LIYV RNA 2 were detectable for both at 24 h p.i. This was likely residual inocula, as the RNA 1 Rep⫺ mutant was unable to replicate and therefore could not sup-

port RNA 2. However, by 48 and 72 h p.i., LIYV RNA 2 was clearly detectable only in cells coinoculated with P32⫺ but not with Rep⫺, indicating that this is progeny RNA 2. These comparative analyses showed that the mutation introduced into the LIYV RNA 1 ORF 2 (P32⫺) did not affect LIYV RNA 1 accumulation, but severely affected the trans replication or accumulation of both positive- and negative-sense LIYV RNA 2. In order to further examine the role(s) of LIYV RNA 1 ORF 2 and/or its encoded P32 in RNA accumulation, two additional LIYV RNA 1 ORF 2 mutants (P32 F1 and P32 F2; Fig. 1 and Table 1) were generated. These mutants had frameshift mutations and inserted stop codons within the RNA 1 ORF 2 coding sequence, and these were located distant from the P32⫺ mutation. Stop codons were introduced at P32 residues 174 and 237 for mutants P32 F1 and P32 F2, respectively. When transcripts of these mutants were coinoculated into protoplasts with wild-type RNA 2, the RNA 1 mutants accumulated to high levels (Fig. 4A). The mutant P32 F1 was not different from wild-type RNA 1 or the P32⫺ mutant, while P32 F2 showed slightly lower accumulation than did wild-type RNA 1 in two of three experiments. However, in three experiments, no LIYV RNA 2 was detected, suggesting that like for the P32⫺ mutant, P32 F1 and P32 F2 do not support wild-type levels of RNA 2 accumulation (Fig. 4B). DISCUSSION The data presented here show several interesting aspects regarding replication and accumulation of LIYV RNAs. First,

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FIG. 4. Accumulation of LIYV RNA 1 mutant and wild-type RNAs. Transcripts of wild-type LIYV RNA 1 (from pSP9/55) and of the Rep⫺, P32⫺, P32 F1, and P32 F2 RNA 1 mutants were separately coinoculated with wild-type LIYV RNA 2 (pSP6) transcripts to 1.2 ⫻ 106 tobacco protoplasts, and 1.2 ⫻ 105 protoplasts were collected at 0, 24, 48, and 72 h p.i. (no protoplasts inoculated with P32 F1 and P32 F2 transcripts were collected at the 48-h time point). Total RNAs purified from 1.2 ⫻ 104 protoplasts collected at the time points indicated were used for Northern blot hybridization with RNA 1 (A) and RNA 2 (B) negative-sense DIG-labeled probes. G indicates the position of LIYV genomic RNAs, and SG indicates the LIYV RNA 1 ORF 2 (P32) subgenomic RNA. Numbers at left correspond to positions of marker RNAs (sizes in nucleotides) analyzed in the same gel. Images were arranged and labeled using Adobe Photoshop 4.0.

accumulation of LIYV genomic RNAs 1 and 2 is not synchronous. LIYV RNA 1 RNAs (both positive- and negative-sense genomic RNAs, as well as the RNA 1, ORF 2 [P32] subgenomic RNA) were always detected very early after inoculation (ca. 12 h p.i.), and the maximal rate of increase for these RNAs occurred between 12 and 24 h p.i. In contrast, our data showed that significant accumulation of positive- and negativesense RNA 2 was only detected between 24 and 36 h p.i. When hybridization signals were compared over time, the amount of LIYV RNA 1 detected at 12 h p.i. was already much greater than the input (inoculum at 0 h p.i.). Conversely, the LIYV RNA 2 signal was not greater than that for inoculum until 36 h p.i. These results were consistently obtained. Also, as inocula contained essentially equimolar amounts of RNAs 1 and 2, this was not due to differing amounts of these RNAs in the inocula. The above data suggest that there may be cis-preferential replication of LIYV RNA 1. LIYV RNA 1 encodes proteins for replication, and the ORFs 1A- and 1B-encoded proteins, most likely translated from the LIYV genomic RNA 1, likely serve to replicate LIYV RNA 1 and direct transcription of the RNA 1 ORF 2 (P32) subgenomic RNA. The P32 subgenomic RNA not only appears quickly after inoculation, but it is the most abundant of all of the LIYV RNAs detected by us in LIYV-infected cells. Efficient replication or accumulation of LIYV RNA 2 does not simultaneously occur with that of LIYV RNA 1. RNA 2 is replicated in trans, and progeny begin to appear only after sufficient accumulation of LIYV RNA 1 (and most likely its encoded proteins). All of our LIYV RNA 2 mutants, when coinoculated with wild-type RNA 1, accumulated to levels essentially equal to

those of wild-type RNA 2 and did not affect the level of LIYV RNA 1 accumulation. Furthermore, during LIYV replication, subgenomic RNAs are generated for downstream ORFs for both RNAs 1 and 2 (20), and the LIYV RNA 2 mutations analyzed here did not affect accumulation of subgenomic RNAs for the RNA 1 ORF 2 (P32) or the RNA 2 HSP70 homolog and P26 ORFs, the subgenomic RNAs examined by us here. This is not so surprising, as previous work from our laboratory (11) showed that LIYV RNA 1 is replication competent in the absence of LIYV RNA 2. In addition, although we evaluated mutations in five of the seven RNA 2 ORFs, we did not mutate the RNA 2 ORFs encoding P5 or P9. However, evidence also suggests that these ORFs (or their encoded proteins) are unlikely to be involved in LIYV RNA accumulation. First, RNA 1 alone accumulates to similar levels with or without coinfection of RNA 2 (11; Fig. 3A). Second, when RNA 1 was coinoculated into protoplasts with LIYV RNA 2 defective RNAs (D RNAs) lacking the P5 and/or P9 ORFs, no effects were seen for LIYV RNA accumulation (20). Taken together, these data suggest that LIYV RNA 2 and its encoded proteins do not significantly affect LIYV RNA accumulation. However, some evidence suggests that in addition to ORFs 1A and 1B, other ORFs may encode proteins that affect RNA accumulation for viruses in the family Closteroviridae. For the Closterovirus BYV, the 3⬘-most ORF on the monopartite genomic RNA has been identified as a replication enhancer (17). The similarly positioned ORF for LIYV is the 3⬘-most ORF on RNA 2 (encoding P26). All members of the family Closteroviridae have similarly positioned ORFs encoding proteins of similar size (e.g., the BYV protein is P21) (1, 6).

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Computer-assisted analysis showed that the LIYV-encoded P26 showed no significant similarity with the BYV-encoded P21, and our data do not suggest that the RNA 2 3⬘-most ORF encodes any sort of replication enhancer affecting LIYV RNA 1 or RNA 2, or even accumulation of the subgenomic RNAs examined here. Interestingly, our data show that mutations in the 3⬘-most ORF of LIYV RNA 1 affect the trans replication and accumulation of LIYV RNA 2. Our data showed that the LIYV RNA 1 ORF 2 mutants were capable of independent replication similar to that of wild-type RNA 1, demonstrating that P32 is not necessary for RNA 1 replication. However, the LIYV P32 mutants were unable to efficiently direct replication and accumulation of LIYV RNA 2. Also, because we constructed three separate mutants for ORF 2 in distinct coding regions of this ORF and obtained similar phenotypes, these data suggest that the ORF 2-encoded P32 is the likely trans enhancer of LIYV RNA 2 accumulation. Replication enhancers have been reported for several multipartite plant viruses and include the ␥RNA-encoded ␥B of Barley stripe mosaic virus (BSMV; 19), the Cowpea mosaic virus (CPMV) M-RNA-encoded 58-kDa protein (4), the Beet necrotic yellow vein virus (BNYVV)-encoded P14 (8), and the Peanut clump virus (PCV) RNA 1-encoded P15 (9). The BSMV ␥B, the BNYVV P14, and the PCV P15 proteins all belong to a group of cysteine-rich proteins, and P14 shares weak but statistically significant similarity with other nucleic acid binding proteins (8). These cysteine-rich proteins influence or enhance replication for all genomic components of their respective virus. In contrast, the CPMV 58-kDa protein is a template-selective replication enhancer. The CPMV 58-kDa protein is needed for efficient replication of the CPMV MRNA but not the CPMV B-RNA; thus, it is a cis replication enhancer. Interestingly, a RNA sequence located within Red clover necrotic mosaic virus (RCNMV) genomic RNA 2 has recently been identified as a transcriptional enhancer, functioning in trans for the synthesis of an RCNMV RNA 1 subgenomic RNA (22). LIYV RNA replication-accumulation kinetics and the RNA 1-mediated trans replication enhancer activity affecting accumulation of LIYV RNA 2 so far appear to be unusual among RNA plant viruses. In this regard, it is interesting to note the lack of nucleotide sequence homology seen for the LIYV genomic RNAs. Only the 5⬘-most five nucleotides and a 23nucleotide sequence near the 5⬘ termini of both LIYV RNAs 1 and 2 are homologous for LIYV RNAs 1 and 2 (12). In contrast, most multipartite RNA plant viruses have conserved or shared 3⬘ nucleotide sequences on the genomic RNA segments for a given virus (5). No complete nucleotide sequences are yet available for other members in the genus Crinivirus; therefore, whether they exhibit sequence characteristics like those of the LIYV genomic RNAs is presently unknown. Presumably, other viruses in this genus would encode a protein like the LIYV P32. The monopartite viruses in the genus Closterovirus do not. ACKNOWLEDGMENTS This work was supported in part by grants from the USDA NRICGP to B.W.F. and a University of California Block Grant and JastroShields Fellowship to H.-H.Y. L.R. was supported in part by a postdoctoral fellowship from Ministerio de Educacio ´n y Ciencia, Spain. B.C. was supported in part by a U. C. Davis President’s Undergraduate Fellowship. REFERENCES 1. Agranovsky, A. A. 1996. Principles of molecular organization, expression, and evolution of closteroviruses: over the barriers. Adv. Virus Res. 47:119– 158.

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