The RNA of maize chlorotic mottle virus - the essential virus in maize lethal necrosis disease - is translated via a panicum mosaic virus-like cap-independent translation element

Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), an emerging disease worldwide that often causes catastrophic yield loss. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that, like other tombusvirids, MCMV RNA contains a cap-independent translation element (CITE) in its 3’ untranslated region (UTR). The MCMV 3’ CITE (MTE) was mapped to nucleotides 4164-4333 in the genomic RNA. SHAPE probing revealed that the MTE is a variant of the panicum mosaic virus-like 3’ CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of a m7GpppN cap structure, which is normally required for eIF4E to bind RNA. The MTE interaction with eIF4E suggests eIF4E may be a soft target for engineered resistance to MCMV. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5’ UTRs of both genomic RNA and the 3’-coterminal subgenomic RNA1 via long-distance kissing stem-loop base pairing to facilitate translation in wheat germ extract and in protoplasts. However, the MTE is a relatively weak stimulator of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs. Most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. We propose that MCMV, and some other positive strand RNA viruses, favors a weak translation element to allow highly efficient viral RNA synthesis. Author Summary In recent years, maize lethal necrosis disease has caused massive crop losses in East Africa and Ecuador. It has also emerged in East Asia. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer full immunity to MCMV. In order to design better resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression required for infection by all positive strand RNA viruses. We identified a structure (cap-independent translation element) in the 3’ untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor in a way that differs from host mRNA interactions with the translational machinery. This difference may guide engineering of – or breeding for – resistance to MCMV. Moreover, this work adds to the diversity of known eukaryotic translation initiation mechanisms, as it provides more information on mRNA structural features that permit noncanonical interaction with a translation factor. Finally, owing to the conflict between ribosomes translating and viral replicase copying viral RNA, we propose that MCMV has evolved a relatively weak translation element in order to permit highly efficient RNA synthesis, and that this replication-translation trade-off may apply to other positive strand RNA viruses.


64
Maize lethal necrosis disease (MLND, also referred to as corn lethal necrosis) first identified in 65 the Americas in the 1970's [1], has recently spread worldwide, causing devastating crop losses 66 and food insecurity across East Africa, where maize is the most important subsistence and cash 67 crop [2][3][4][5][6][7][8][9]. It has also emerged in China [10], Taiwan [11], Spain [12], and Ecuador, where the 68 damage was so catastrophic in 2015 and 2016 that a state of emergency was declared [13,14] Mapping the 3'-cap independent translation element in MCMV 119 To roughly map the 3' CITE of MCMV, we translated 3'-truncated transcripts from a full-length 120 cDNA clone of the MCMV genome (pMCM41 [29]). pMCM41 DNA templates were transcribed 121 in the absence of cap analog while pMCM721, which lacks the 5' terminal A of the MCMV 122 genome, was used for capped transcripts, because the 5' A of pMCM41 (identical to MCMV 123 RNA) cannot be capped using an m 7 GpppA cap analog and T7 RNA polymerase (KS 124 unpublished observation). Plasmid psgRNA1 was the template for transcription of full-length 125 sgRNA1, the mRNA for the 25 kDa CP and the movement proteins [28]. Transcribed RNAs and 126 RNA isolated from virions (vRNA) were translated in wheat germ extract (WGE) in the presence 127 of 35 S-methionine. The full-length, infectious transcript from SmaI-linearized pMCM41 and vRNA 128 yielded two protein products, P32 and P50 from the 5'-proximal overlapping ORFs ( Fig 1B). 129 Interestingly, vRNA yielded much less P32 protein, relative to P50, than did the transcribed 130 mRNA. Also, a faint band comigrating with CP is visible from both vRNA and the full-length 131 transcript. The expected 111 kDa protein generated by readthrough of P50 stop codon was not 132 detected, most likely because ribosomal readthrough occurs at a very low rate in these 133 conditions. Readthrough products have been difficult to detect among the in vitro translation 134 products of other tombusvirid genomes as well [44][45][46]. Unlike the full-length genomic RNA from 135 SmaI-cut pMCM41, which yielded substantial protein products, the uncapped 3'-truncated 136 transcripts produced almost no detectable protein product, suggesting that the 3' CITE is 137 downstream of the Spe I site at nt 4191 ( Fig 1B). It is noteworthy that translation in the 138 presence of a 5' cap on full-length and truncated pMCM41-derived RNAs gave much more 139 translation product than uncapped full-length pMCM41 transcript or vRNA, indicating that the 140 viral genome may be a relatively inefficient mRNA. 141 To rapidly map the 3' CITE location at high resolution, a luciferase reporter (MlucM) was 142 constructed such that the coding region of the virus was replaced by the firefly luciferase (Fluc) 143 coding sequence (Fig 2A). Deletion analyses showed little decrease in translation in vitro or in vivo when either the 3'-terminal 104 nt (nts 4334-4437) or the first 169 nt (nts 4095-4263) of the 145 3' UTR were deleted (Fig 2B). Additional constructs that included the adjacent sequence 146 upstream of the 3' UTR, up to nt 3578 in the CP ORF, translated more efficiently than those that 147 contained only the 3' UTR (S1 Fig). However, the sequence upstream of the 3' UTR (3578-148 4108) alone was not enough to support translation, and the greatest contributor to translation 149 was mapped to the 3' UTR. Numerous deletions in the MCMV 3' UTR revealed that the region 150 between nucleotides 4164-4333 produced luciferase activity >100% of that from the full-length 151 3' UTR in vitro and about 50% in vivo. The lower level of translation in vivo may be due to 152 reduced RNA stability owing to the absence of the 3' end, which is thought to confer stability in 153 related viruses because of its highly base-paired terminal bases [47][48][49]. Deletions within nts 154 4164 to 4333, especially of nts 4200-4300, reduced luciferase translation in vitro, so in 155 subsequent studies we focused on nts 4164-4333 to characterize the MCMV 3' CITE (MTE). 156

157
To determine the structure of the MTE, we first attempted to predict its secondary structure 158 computationally. Two different algorithms, MFOLD [50], and ViennaRNA Package [51], 159 predicted a hammer-shaped structure unlike any known 3' CITE, so we proceeded to determine 160 its secondary structure experimentally by subjecting the MTE (nts 4164-4333) to selective 2'-161 hydroxyl acylation analyzed by primer extension (SHAPE) probing ( Fig 3A). This revealed that 162 the MTE consists of a long helix with various asymmetric internal loops topped by two branching 163 stem loops (Fig 3B), which differed from the computer-predicted structure. The main stem 164 contains a purine-rich bulge between nucleotides 4216-4223. In the presence of magnesium 165 ion, bases G4215, A4216, and G4219 were hypermodified by the SHAPE reagent benzoyl cyanide, 166 while bases AGA4221-4223 became hypomodified (Fig 3A). The MTE also contains a single-167 stranded "bridging domain" (nts 4246-4250) connecting the two branching stem-loops, which 168 was moderately modified in the presence and absence of magnesium. Side loop-I (SL-I4235:4241) houses a pentamer, UGCCA4236-4240, in its loop that is complementary to sequence UGGCA in 170 the 5'-UTR. These pentamers may create a long-distance base-pairing interaction between the 171 3' and 5' UTRs (discussed later  4A). This alignment revealed a consensus structure 179 with more variability than reported previously [53], because more predicted PTE sequences are 180 aligned than previously. The MTE and PTEs contain a purine-rich bulge with at least one highly 181 conserved G. However, the previously termed "C-rich" domain of PTE that bridges between 182 stem-loops 1 and 2 is not always C-rich, thus we now call it the bridging domain. One putative 183 PTE, from Pea stem necrosis virus (PSNV), contains no bridging domain and only a two base-184 pair stem in stem-loop 2 ( Fig 4A). However, it has not been demonstrated to be functional. 185 Potential pseudoknot base pairing between the purine-rich bulge and the bridging domain 186 (square brackets Fig 4A)  have little, if any pseudoknot base pairing ( Fig 3B and S2 Fig, respectively). However, 213 presence of a strong pseudoknot does not guarantee a strong translation enhancer, as 214 indicated by PEMV2 and HCRSV PTEs ( Fig 4A, Fig 4C, S1 Fig, S2 Fig). The role of potential 215 pseudoknot base pairing is explored further below. 216 We constructed a series of mutations in the purine-rich bulge and bridging domain to test 217 whether changes in these areas predicted to strengthen or weaken the pseudoknot had effects 218 on translation efficiency (Fig 5). These included mutations designed to determine if a stronger pseudoknot could increase translation activity. Mutant A4248U, which should lengthen the 220 proposed wild type pseudoknot from two (AG4221-4222:CU4249-4250) to three (AGA4221-4223:UCU4248-221 4250) base pairs, translated only 55% as efficiently as wild type in WGE (Fig 5B). This mutant 222 could also potentially form an ACU4216-4218:AGU4246-4248 pseudoknot helix. To disrupt that 223 possibility, a U4218A mutation was added. This double mutant translated 70% as efficiently as 224 wild type in WGE ( Fig 5C). However, neither of these mutants translated appreciably in the 225 more competitive conditions in protoplasts. In other constructs, mutations in both the purine-rich 226 bulge and the bridging domain were introduced to generate pseudoknot base pairing predicted 227 to be more stable than wild type. In constructs in which the purine-rich bulge remained purine- the single-domain mutants. The GGG:CCC pseudoknot actually yielded 50% more luciferase 232 than wild type in WGE and protoplasts (Fig. 5F), whereas the AAAA:UUUU predicted 233 pseudoknot translated 35% as efficiently as wild type in WGE (Fig 5I), which was slightly greater 234 than the UUU mutation alone (which may form a weak pseudoknot containing two G:U pairs) or 235 the AAA mutation in the purine-rich bulge. Each of these mutants translated about 15-20% as 236 efficiently as wild type in WGE. However, none of this set of mutants translated detectably in 237 protoplasts (Fig 5, panels G, H, I). Swapping the purines and pyrimidines to create a potential: 238 ACCC:GGGU pseudoknot helix gave low and no cap-independent translation in WGE and 239 protoplasts, respectively ( Fig 5J). One mutation, G4219U in the purine-rich bulge was not 240 predicted to affect pseudoknot interactions and did not affect translation activity of the MTE (Fig  241   5K). This is interesting because G4219 is hypermodified in the presence of Mg 2+ (Fig 3). 242 Overall, with one rather modest exception (Fig 5F), mutations designed to increase pseudoknot 243 base pairing altered the structure in such a way as to decrease translation efficiency.  We next tested the ability of mutant MTEs to bind eIF4E, in order to determine if eIF4E 266 binding correlates with the translation enhancement function, as was observed previously for 267 the TPAV PTE. Purine-rich bulge mutants U4218G and G4219U, which gave 50% and 100% of 268 wild type translation, respectively, showed very similar EMSA profiles to wild type MTE (Fig 6). 269 The mutant designed to strengthen the pseudoknot interaction, U4218G/GA4247-4248CC, and which gave 150% of wild type translation ( Fig 5F) shifted similarly to U4218G alone, but with 271 slightly less complete binding. This may be due to partial misfolding, as detected in the full- the P32 (AUG118-120) and P50 (AUG137-139) ORF start codons, was subjected to SHAPE probing 289 to determine which region was most likely to be available (single stranded) to interact with the 290 MTE ( Fig 7A). The 5' UTR (nts 1-117) was found to consist of a large stem-loop with several 291 large bulges, followed by a short stable stem-loop terminating 5 nt upstream of the start codon 292 ( Fig 7B). The first potential MTE-interacting sequence (GGCA12-15) is buried in a stem helix, 293 while the UGGCA103-107 is in a favorable loop ( Fig 7B). This led us to suspect that UGGCA103-107 294 is the potential base pairing sequence that interacts with MTE side loop 1. 295 Long distance base pairing of the MTE to the 5' UTR 296 We next defined functionally which (if any) of the above candidate sequences base pairs to the 297 MTE. Mutations were introduced in the XGCCA regions in the 5' UTR (nts 11-15 or 103-107) 298 and the MTE UGCCA4236-4240 region ( Fig 8A). Mutation of G13 to C caused only a small 299 decrease in luciferase activity in WGE and in oat protoplast translation systems ( Fig 8B). In 300 contrast, mutation of G105 to C, reduced luciferase activity by ~75% in WGE and protoplasts. The MTE should also base pair to the 5' UTR of sgRNA1, to allow translation of the viral 315 coat and movement proteins. Indeed, we identified a sequence, UGGCA2979-2983 in the short 25 316 nt 5' UTR of sgRNA1, which matches the UGGCA103-107 that base pairs to the MTE. This 317 sequence is predicted to be in the terminal loop of the stem-loop that occupies the 5' UTR of 318 sgRNA1 (Fig 9A), which starts at nt 2971 [28]. We investigated both the effect of this short 25 nt 319 5' UTR on MTE-mediated translation efficiency, and the role of base pairing (if any) between UGGCA2979-2983 and UGCCA4236-4240 in the MTE. In WGE, the sgRNA1 5' UTR enabled 321 translation about equally efficiently as did the genomic 5' UTR, while translation was about two-322 thirds as efficient in oat protoplasts (Fig 9B), perhaps due to less RNA stability conferred by the 323 shorter 5' UTR. Separate G2981C and C4238G mutations in the sgRNA1 5' UTR and the MTE, 324 respectively, reduced translation significantly in both WGE and oat protoplasts. The double 325 mutant, designed to restore predicted base pairing, with G2981C and C4238G in the same 326 construct, gave surprising results. In WGE, as predicted, the double mutant fully restored 327 translation to wild type levels. However, in oat protoplasts, the same mRNA was as 328 nonfunctional as those containing single G2981C and C4238G mutations, showing no 329 restoration of translation whatsoever. Because WGE is a high-fidelity translation system that 330 measures only translation, independent of the complicated environment of the cell, we conclude 331 that the long-distance base pairing is necessary for efficient cap-independent translation. We 332 speculate that the G2981C mutation altered the RNA in such a way as to make it highly 333 unstable in cells or able to fortuitously interact with cellular components that preclude 334 translation, and which are absent in WGE. 335

336
To determine the effects of mutations that affect translation in the natural context of genomic 337 RNA, selected mutations from the luciferase experiments were introduced into the MCMV 338 infectious clone pMCM41. Firstly, uncapped, full-length genomic RNA transcripts from pMCM41 339 mutants were translated in WGE, and the predominant 35 S-met-labeled viral protein products 340 (P50, P32 and P25) were observed. In agreement with the luciferase reporter constructs, 341 mutants MCM41C4238G and MCM41G105C, which disrupt the long-distance base pairing between 342 MTE and 5' UTR, yielded less viral protein than wild type (Fig 10A). In contrast, the double 343 mutant, MCM41G105C/C4238G, which restores the long-distance base pairing, translated more 344 efficiently than wild type RNA for the P32 and P50 proteins. A 25 kDa protein, presumably the viral coat protein (MW 25 kDa), was not expected to be translated much from genomic RNA as 346 seen in Fig 1B, but it appeared in this experiment. Its translation remained at about the same 347 reduced level in the double mutant as in the single mutants. MCM41 mutants G4219U, 348 U4218G, and Δ4200-4300 translated similarly to the luciferase constructs, relative to wild type. 349 However, the mutant designed to form a GGG:CCC pseudoknot in the MTE, 350 MCM41U4218G/GA4247-4248CC, showed a substantial decrease in translation, in contrast to the same 351 mutation in the luciferase reporter, in which translation was increased by 50% (Fig 5F). 352 To determine if the mutants that translated poorly in the full-length genome context did   Table 1). The few 393 single mutants (C4238G, G105C, U4218G) that showed symptoms at 14 dpi had reverted to 394 wild type MCM41 (Table 1). MCM41G4219U had a similar infectivity to wild type, but in about half of the infected plants the virus reverted to wild type (Table 1). Moreover, the MCM41G4219U that 396 did not revert to wild type accumulated less RNA (Fig. 10C), suggesting that, although the 397 G4219U mutation is tolerated, the wild type sequence is more competitive. With one exception 398 (below) MCM41G105C/C4238G retained its introduced mutations but the infectivity (Table 1) and 399 RNA accumulation (Fig 10C) also was reduced relative to wild type. from 289 aa to 125 aa, and changed amino acid 119 from valine to glutamic acid in the P50 and 405 P111 (RdRp) proteins. This spontaneous mutant did not induce symptoms until 12 dpi, but after 406 14 dpi, symptoms were more extreme than wild type, giving nearly translucent leaves (Fig 10D). 407 In summary, the MCMV genome tolerated few mutations, and only those mutations that allowed 408 the most efficient translation replicated in maize plants. 409 Previous studies of nine PTEs revealed a common secondary RNA structure composed of a 424 branched structure with two side loops (Fig 4) connected by a pyrimidine-rich bridging domain 425  The MCM41C105G/G4238C transcript, which translates more efficiently than wild type in 462 WGE, replicates indistinguishably from wild type MCM41 in protoplasts, but accumulates to 463 much lower levels in whole plants. This is to be expected because the G4238C mutation in the 464 MTE would prevent base pairing to the 5' end of sgRNA1, which remains a wild type sequence. reporter constructs revealed no striking difference in translation efficiency conferred by the 5' 474 UTR (Fig 9). This is interesting, because the secondary structure and length of the 5' UTR is 475 much less in the sgRNA1 5' UTR (24 nt, DG = -6.6 kcal/mol) compared to that of genomic RNA

Efficient replication vs efficient translation?
500 It is possible that the relatively weak stimulation of translation by the MTE, and the relatively 501 inefficient translation of viral genomic RNA, relative to artificially capped genomic RNA (Fig 1B), translate "poorly" in order to make it available to the viral replicase for highly efficient RNA 508 replication. Moreover, owing to its abundance, genomic RNA may simply not need to translate 509 efficiently. Indeed, highly efficient translation of such an abundant RNA could overwhelm the 510 ribosomes, preventing translation of host mRNAs, harming the cell, and thus the virus itself. 511 The 5' UTR of the unrelated wheat yellow mosaic bymovirus harbors an IRES in a dynamic 512 equilibrium state, which also gives suboptimal translation [86], perhaps also to allow efficient 513 replication. In contrast, BYDV RNA has a highly efficient 3' CITE (a BTE) [87], but accumulates 514 to much lower levels in cells (not detectable by staining of total RNA). BYDV may have adopted 515 a strategy to accumulate low levels of RNA and virus particles, which are adapted to very 516 efficient acquisition by aphids. This RNA would require much more efficient translation in order 517 to compete with host mRNAs for ribosomes. Thus, viruses such as MCMV are "replication 518 strategists", going "all in" on RNA synthesis at the expense of translation efficiency, while 519 others, such as BYDV are "translation strategists", translating efficiently at a cost to RNA 520 replication.

522
As mentioned above, understanding translation may lead to strategies for resistance. The MTE 523 binds eIF4E via what must include different molecular contacts than the binding of a 5' m 7 G cap 524 to eIF4E via the cap-binding pocket [88]. Thus, it may be possible to identify mutants of eIF4E 525 that lose the ability to bind the MTE but retain a functional cap-binding pocket to allow 526 translation of host (capped) mRNAs. In melon, such a resistance mechanism has been 527 The full length infectious clone of the MCMV genome (pMCM41) was described previously [29]. 540 For psgRNA1 construction, template DNA pMCM41 was used to amplify MCMV nt 2972-4437 541 using Vent DNA polymerase, and oligonucleotides 3'SmaI (5'-agcaagcttcccGGGCCGGAAGAG 542 [29] and sgRNA1 (5'GGTATTTTGGCAGAAATTCC) that were phosphorylated with T4 543 polynucleotide kinase. The vector pT7E19 [92] was digested with Sac I followed by mung bean 544 nuclease digestion. Vector and insert were digested with Hind III, phenol/chloroform extracted, and precipitated prior to ligation with T4 DNA ligase. DNA was added to competent E. coli 546 DH5α, selected on ampicillin/XGal plates, and screened by restriction digests and sequencing. 547 Transcripts made from psgRNA1 linearized with SmaI contain MCMV nt 2971-4437, the 548 complete sgRNA1. All enzymes were from New England Biolabs. MlucM was constructed using 549 Gibson Assembly kit (New England Biolabs) such that a firefly luciferase (luc2, Promega) 550 reporter gene was flanked by the 5' and 3' UTRs of MCMV (Fig 1). A Q5 Site-directed 551 mutagenesis kit (New England Biolabs) with custom forward and reverse primers (S1 Table) 552 was used to generate the deletions (Fig 2) and mutants (Figs 5, 8, 9) 1), plasmid DNA templates were linearized 557 by restriction digestion or amplified by PCR to ensure correct template length. The RNAs were 558 synthesized by in vitro transcription with T7 polymerase using MEGAscript (for uncapped RNAs) 559 or mMESSAGE mMACHINE (for capped RNAs) kits (Ambion). RNAs used as probes for 560 uncapped EMSA assays were generated using MEGAshortscript kit (Ambion). RNA transcripts 561 were purified according to manufacturer's instructions, RNA clean and concentrator kit (Zymo 562 Research) was used for non-radioactive RNA preparation. RNase-free Bio-Spin columns P-30 563 (Bio-Rad) were used for radiolabeled RNA. RNA integrity was verified by 0.8% agarose gel 564 electrophoresis. RNA concentration was determined by spectrophotometry for non-radiolabeled 565 RNA. Radiolabeled RNA concentration was calculated by measuring the amount of incorporated 566 radioisotope using a scintillation counter. At Oklahoma State University (Fig 1) full length (nt 1-567 4437) or 3'-truncated (nt 1-4195) genomic RNA was synthesized with T7 RNA polymerase (New 568 England Biolab) and either pMCM41 or pMCM721 linearized with Sma I or Spe I following 569 company protocols for uncapped or capped RNA synthesis. Unincorporated NTPs were removed by three rounds of ammonium acetate/ethanol precipitation and resuspension in 571 nuclease-free water. pMCM721 contains a G residue between the T7 promoter and MCMV 572 sequence, allowing synthesis of capped or uncapped RNAs 1 nt longer than WT [29]. The 3K 573 MCMV RNA transcript templates were made by PCR using primers p9KO2 574 Inhibitor (Invitrogen), and 20mM Tris-HCl/10% glycerol for 25 minutes in ice. Then, 3 μL of 50% 640 glycerol was added to each reaction. Immediately after, 7μL of RNA-protein mixture were 641 loaded into a 5% polyacrylamide (acrylamide:bis-acrylamide 19:1), Tris-borate/EDTA (TBE) gel, 642 which was run at ~4°C at 110V for 45 minutes in 0.5X cold TBE buffer. Gels were dried on 643 Whatman 3MM paper and exposed to a phosphorimager screen overnight. Phosphor screens 644 were scanned in a Bio-Rad PhosphorImager, and radioactivity counts were analyzed using Quantity One software (Bio-Rad). Statistical analysis was performed using GraphPad Prism 646 software. 647

660
Maize (B73 and sweet corn varieties) was grown in growth chambers on a 16:8 photoperiod 661 with a temperature setting of 25°C/22°C (day/night). Maize seedlings were inoculated at the 662 three-leaf stage. Plants were dusted with carborundum and inoculated on the third leaf with 10 663 μg of purified MCMV transcripts in 10mM sodium acetate, 5mM calcium chloride, and 0.5% 664 bentonite by stroking three times with a freshly-gloved finger. Inoculated leaves were rinsed 665 with water 10 minutes post inoculation; rinsed water was collected and autoclaved prior to 666 disposal. Total plant RNA was extracted using Trizol (Invitrogen) from 100 mg of the newest 667 systemic leaves for northern blot hybridization (14 dpi) and cDNA synthesis. Quality of RNA was 668 evaluated in 0.8% native agarose gel, and RNA was quantified using a Nanodrop 669 Spectrophotometer. RNA extracted from infected plants was subjected to cDNA synthesis and RT-PCR and sent to the Iowa State University DNA Facility for sequencing to evaluate the 671 presence of introduced mutations. 672

673
RNA isolated from BMS protoplasts had higher ratio of degraded RNA genomic/ sub-genomic 674 RNA in northern blot hybridizations such that lower molecular weight fragments overpowered 675 the genomics and sub-genomics RNAs. However, a trend on the amount of MCMV RNA 676 detected suggested that replication was still occurring in protoplast; thus, dot blots were used 677 instead. Unfractionated RNA from protoplasts was denatured prior to immobilization on a nylon 678 membrane using a vacuum manifold apparatus as described in Brown et. al. 2004 [98]. RNA 679 from maize plants was not degraded so it was subjected to northern blot hybridization. Total 680 RNA from 100 mg newest leaf samples (14 dpi) was denatured in a formaldehyde/formamide 681 buffer solution by heating at 65°C for 15 minutes, follow by separation in a 0.8% denaturing 682 agarose gel and transferred to a nylon membrane. Both dot blot and northern blot nylon 683 membranes were UV cross-linked, follow by prehybridization (50% formamide, 5X SSC, 200 684 μg/mL polyanetholesulfonic acid, 0.1% SDS, 20mM sodium phosphate, pH 6.5) at 55°C for 2 685 hours. Membranes were hybridized using 32 P-labeled probe complementary to MCMV nts 3811-686 4356, which had been transcribed using SP6 RNA polymerase. Washed blots were wrapped in 687 plastic and exposed to phosphor screens. Phosphor screens were scanned in a Bio-Rad 688 PhosphorImager, radioactivity counts were analyzed using Quantity One software (Bio-Rad). 689 Statistical analysis was performed using GraphPad Prism software. 690