JOURNAL OF VIROLOGY, Mar. 2010, p. 2623–2628 0022-538X/10/$12.00 doi:10.1128/JVI.02142-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 5
Stability of Murine Cytomegalovirus Genome after In Vitro and In Vivo Passage䌤 Tammy P. Cheng,1 Mark C. Valentine,2 Jian Gao,1 Jeanette T. Pingel,2 and Wayne M. Yokoyama1,2* Division of Rheumatology, Department of Medicine, Washington University School of Medicine,1 and Howard Hughes Medical Institute,2 St. Louis, Missouri 63110 Received 9 October 2009/Accepted 4 December 2009
While large DNA viruses are thought to have low mutation rates, only a small fraction of their genomes have been analyzed at the single-nucleotide level. Here, we defined the genetic stability of murine cytomegalovirus (MCMV) by whole-genome sequencing. Independently assembled sequences of three sister plaques showed only two single-base-pair substitutions after in vitro passage. In vivo-passaged MCMV likewise demonstrated low mutation rates, comparable to those after in vitro passage, indicating high genome stability of MCMV at the single-nucleotide level in the absence of obvious selection pressure. sister plaques were selected and independently amplified in vitro by sequential passages for 21 days in NIH-3T12 cells. Virions were then isolated from the culture medium, and genomic DNA was extracted for shotgun sequencing by the Sanger method. Using the bioinformatics software package Phred/Phrap/Consed (9), we then independently assembled complete genomic sequences of these clones, named MCMVWT1, -WT2, and -WT3, with an average of 8-fold coverage per genome. Moreover, we subsequently validated these sequences (see below). Interestingly, the sequences from MCMV-WT1 and -WT2 were identical, whereas MCMV-WT3 differed at only two base pairs (bp): a G3A change at residue 8847 and an A3G change at residue 227424. The first change results in a synonymous mutation in putative ORF m09, while the second change does not fall into any known or predicted ORF. Assuming that these mutations do not affect viral growth, we estimated the mutation rate for MCMV after in vitro passage to be ⬃1.4 ⫻ 10⫺7 mutations per bp per day (2 mutations in 3 genomes at 230,379 bp per genome per 21 days). Thus, the MCMV genome is highly stable at the single-nucleotide level during multiple rounds of in vitro passage. In contrast to the near identity between the sister plaques, our laboratory’s Smith strain MCMV differed from the previously published Smith strain (NCBI accession number U68299) (14)
Large DNA viruses, such as herpesviruses, are thought to have low mutation rates as estimated by methods such as analysis of restriction fragment length polymorphisms or function of individual genes (10, 16). However, these analyses sample only a small fraction of the genome (11, 15). Moreover, in the presence of selective pressure, mutations have been identified in both human and murine cytomegalovirus (HCMV and MCMV, respectively) (5, 7, 17). For example, in HCMV, mutations in UL97 account for ganciclovir (GCV) resistance in up to 25% of immunosuppressed patients infected with HCMV (12, 18). However, whether these resistant mutant strains arise de novo or represent new infection is impossible to ascertain in the clinical setting. Previous studies demonstrated that after in vivo passage, MCMV does acquire de novo mutations. Mutants emerge after passage through mice lacking adaptive immunity but carrying the Cmv1r allele, which encodes the Ly49H activation receptor on NK cells (7, 19). The only known ligand for Ly49H is MCMVencoded open reading frame (ORF) m157. In mice infected with a plaque-purified MCMV clone containing intact m157, all escape viruses had m157 mutations. These mutants carried either single-amino-acid substitutions or premature stop codons and demonstrated increased virulence in naïve Cmv1r mice (8). In contrast, there were no mutations in the adjacent ORFs, m156 and m158. Taken together, these data indicate that mutations occur in both HCMV and MCMV under selective pressure. However, absent this pressure, their genomewide stability has not been determined at the single-nucleotide level. In the current study, we set out to detail MCMV genomewide sequence changes after in vitro and in vivo passages in the absence of obvious selection pressure. To determine MCMV genome stability in vitro, we subcloned our laboratory stock of Smith strain MCMV (a gift from Herbert Virgin, Washington University, St. Louis, MO) that had been previously passaged in vivo. Our stock was plaque purified twice on NIH-3T12 monolayers. At the second round of plaque purification, three
* Corresponding author. Mailing address: Washington University School of Medicine, Division of Rheumatology, Campus Box 8045, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: (314) 362-9075. Fax: (314) 362-9257. E-mail:
[email protected]. 䌤 Published ahead of print on 16 December 2009.
FIG. 1. Genomic differences between MCMV-WT1 and Smith strain MCMV sequenced by Rawlinson and colleagues. Differences between MCMV-WT1 and the Rawlinson sequence are summed in 100-bp, nonoverlapping intervals along the genomic position. 2623
2624
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J. VIROL. TABLE 1. Predicted ORFs in MCMV-WT1 compared to those in the previously published sequence
ORF
m01 m02 m03 m04 m05 m06 m07 m08 m09 m10 m11 m12 m13 m14 m15 m16 m17 m18 m19 m20 m21 m22 M23 m23.1 M24 M25 m25.1 m25.2 m25.3 m25.4 M26 M27 M28 m29 m29.1 m30 M31 M31b M32 M34 M35 M37 M38 m38.5c m39 m40 m41 m42 M43 M44 m44.1 m44.3 M45e1 M45e2 m45.1 m45.2 M46 m48.1 m48.2 M49 M50 M51 M52 M53 M55 M56
C
C C C C C C C C C C C C C
C C C C C C C C C C C C C C C C C C C C
Rawlinson’s MCMV sequenceb
MCMV-WT1
Stranda Start
End
ORF length
Start
End
ORF length
468 1033 2270 3267 4179 5291 6463 7459 8632 9624 10715 11686 12599 13085 14085 15044 15749 17071 20338 20579 22644 23585 23777 24825 25147 26014 28997 28997 30244 30244 31346 32247 34486 35747 36030 36885 37281 38777 39283 43086 45912 49444 50465 51783 52487 53268 53786 54355 55354 57888 58759 59144 59518 62773 59520 62810 63044 73566 73575 73923 75505 76519 76919 78465 83004 85711
870 2013 3109 4063 5200 6336 7407 8529 9513 10499 11614 12504 13000 13990 15065 15676 16951 20193 20781 23044 23333 23899 24952 25160 26118 28812 30601 30280 31656 31215 31924 34295 35778 36475 36661 39071 39071 39079 41439 45650 47471 50481 51958 52523 53203 53633 54202 54846 57147 59123 60108 59428 62160 62880 63042 62890 63928 73877 73871 75533 76455 77220 78471 79462 85811 88107
402 980 839 796 1,021 1,045 944 1,070 881 875 899 818 401 905 980 632 1,202 3,122 443 2,465 689 314 1,175 335 971 2,798 1,604 1,283 1,412 971 578 2,048 1,292 728 631 2,186 1,790 302 2,156 2,564 1,559 1,037 1,493 740 716 365 416 491 1,793 1,235 1,349 284 2,642 107 3,522 80 884 311 296 1,610 950 701 1,552 997 2,807 2,396
480 999 2236 3270 4185 5300 6463 7459 8632 9624 10715 11686 12599 13085 14085 15044 15749 17071 20338 20802 22645 23586 23778 24826 25148 26015 28998 28998 30245 30245 31347 32247 34486 35747 36109 36884 37279 38775 39280 43083 45909 49441 50462 51780 52484 53265 53783 54352 55351 57885 58756 59141 59515 62769 61764 62806 63040 73562 73571 73919 75501 76515 76915 78461 83003 85716
836 1979 3102 4070 5210 6337 7407 8529 9513 10499 11614 12504 13000 13990 15065 15676 16951 20193 20781 23045 23334 23900 24953 25161 26119 28813 30602 30281 31657 31216 31925 34295 35778 36730 36660 37729 38829 39065 41436 45647 47468 50478 51955 52520 53200 53630 54199 54843 57144 59120 60105 59425 62145 62876 63038 62886 63924 73873 73867 75529 76451 77216 78468 79462 85816 88112
356 980 866 800 1,025 1,037 944 1,070 881 875 899 818 401 905 980 632 1,202 3,122 443 2,243 689 314 1,175 335 971 2,798 1,604 1,283 1,412 971 578 2,048 1,292 983 551 845 1,550 290 2,156 2,564 1,559 1,037 1,493 740 716 365 416 491 1,793 1,235 1,349 284 2,630 107 1,274 80 884 311 296 1,610 950 701 1,553 1,001 2,813 2,396
No. of differencesc
1 15 74 31 3
1
1 1 1 1 1
1 1
1 5 1 2 3 61 1
Continued on following page
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2625
TABLE 1—Continued ORF
m58 m59 M69 m69.1 M70 M71 M72 M73 M73.5e2 m74 M75 M76 M77 M78 M79 M80 M82 M83 M84 M85 M87 M88 m90 M91 M92 M93 M94 M89Ex1 M95 M96 M97 M98 M99 M100 M102 M103 M104 M105 m106 m106.1 m106.3 m107 m108 M114 M115 M116 m117 m117.1 M118 m119.1 m119.2 m119.3 m119.4 m119.5 m120 m120.1 M121 M121 m123.1 m123Ex2 m124 m124.1 m125 m126 m127
C C C C C
C C C C C C
C
C C C C C C C C C C C C C C C C C C C C C C C C
Rawlinson’s MCMV sequenceb
MCMV-WT1
Stranda Start
End
ORF length
Start
End
ORF length
91756 93236 96284 98621 99101 101994 103122 104191 105888 104587 106205 108479 109026 111084 112737 113512 115812 117718 120186 122293 127487 130347 133020 133768 134175 134833 136334 137487 138379 139632 140238 142198 143820 144393 145693 148279 149210 151125 154010 154293 155878 162083 162310 165696 166484 167305 169313 169641 171080 172156 173122 173510 174154 174254 174399 174740 175779 175779 181963 181756 182033 182111 183536 184635 185290
92459 94393 98812 98979 101995 102893 104327 104609 106160 105903 108382 109242 110912 112498 113513 115607 117611 120147 121949 123228 130267 131626 133976 134172 134867 136379 137370 138380 139632 140021 142168 143883 144158 145508 148131 149232 151324 153971 154453 154553 156015 162777 162870 166484 167308 169242 171010 171055 172045 173091 173490 173821 174435 174589 174674 175825 177875 177875 182319 181866 182380 182518 183865 184910 185691
703 1,157 2,528 358 2,894 899 1,205 418 272 1,316 2,177 763 1,886 1,414 776 2,095 1,799 2,429 1,763 935 2,780 1,279 956 404 692 1,546 1,036 893 1,253 389 1,930 1,685 338 1,115 2,438 953 2,114 2,846 443 260 137 694 560 788 824 1,937 1,697 1,414 965 935 368 311 281 335 275 1,085 2,096 2,096 356 110 347 407 329 275 401
91761 93241 96193 98530 99010 101903 103031 104100 105797 104496 106110 108384 108931 110989 112639 113414 115711 117614 120082 122189 127383 130243 132920 133668 134075 134733 136234 137390 138282 139535 140141 142101 143723 144296 145596 148182 149113 151028 153913 154196 155781 161983 162210 165596 166384 167205 169213 169541 170980 172056 173022 173410 174054 174154 174299 174640 175679 175679 181863 181656 181933 182011 183436 184535 185190
92465 94263 98721 98889 101904 102802 104236 104519 106069 105812 108287 109148 110817 112404 113415 115507 117507 120043 121845 123124 130163 131523 133876 134072 134767 136280 137271 138283 139535 139924 142072 143786 144061 145411 148034 149135 151227 153874 154356 154456 155918 162678 162770 166384 167208 169142 170910 170956 171945 172991 173390 173721 174335 174489 174574 175725 177775 177775 182219 181766 182280 182418 183765 184810 185591
704 1,022 2,528 359 2,894 899 1,205 419 272 1,316 2,177 764 1,886 1,415 776 2,093 1,796 2,429 1,763 935 2,780 1,280 956 404 692 1,547 1,037 893 1,253 389 1,931 1,685 338 1,115 2,438 953 2,114 2,846 443 260 137 695 560 788 824 1,937 1,697 1,415 965 935 368 311 281 335 275 1,085 2,096 2,096 356 110 347 407 329 275 401
No. of differencesc
8 5 1 2 5 8 19 30 2 4 2 11 6 13 10 1 2
1 11 1 1
2
1
Continued on following page
2626
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J. VIROL. TABLE 1—Continued
ORF
m128Ex3 m129 m130 m131 m133Ex1 m134 m135 m136 m137 m138 m139 m140 m141 m142 m143 m144 m145 m146 m147 m148 m149 m150 m151 m152 m153 m154 m155 m156 m157 m158 m159 m160 m161 m162 m163 m164 m165 m166 m167 m168 m169 m170
C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
Rawlinson’s MCMV sequenceb
MCMV-WT1
Stranda Start
End
ORF length
Start
End
ORF length
186185 187447 187907 188126 188978 189968 189995 190410 191188 192333 194182 196162 197805 199541 201065 202843 204130 205743 206963 207029 207427 207724 208915 210342 211688 213043 214535 215635 215996 217033 218271 219699 220573 221287 221976 222467 223381 224514 225880 228021 228411 229440
187399 187947 188380 188476 189895 190381 190321 191171 192192 194042 196116 197616 199331 200848 202694 203994 205593 206876 207400 207388 208116 208890 210084 211478 212905 214149 215668 216078 216985 218103 219467 220625 221250 221766 222515 223750 224379 225662 227190 228566 228809 230147
1,214 500 473 350 917 413 326 761 1,004 1,709 1,934 1,454 1,526 1,307 1,629 1,151 1,463 1,133 437 359 689 1,166 1,169 1,136 1,217 1,106 1,133 443 989 1,070 1,196 926 677 479 539 1,283 998 1,148 1,310 545 398 707
186085 187347 187807 188026 188878 189868 189895 190310 191088 192233 194082 196062 197705 199441 200920 202742 204029 205642 206862 206928 207326 207623 208814 210241 211587 212942 214434 215534 215895 216932 218170 219598 220472 221186 221875 222366 223280 224413 225779 227920 228310 229339
187299 187847 188280 188376 189795 190281 190221 191071 192092 193942 196016 197516 199231 200748 202593 203893 205492 206775 207299 207287 208015 208789 209983 211377 212804 214048 215567 215977 216884 218002 219366 220524 221149 221665 222414 223649 224278 225561 227089 228465 228708 230046
1,214 500 473 350 917 413 326 761 1,004 1,709 1,934 1,454 1,526 1,307 1,673 1,151 1,463 1,133 437 359 689 1,166 1,169 1,136 1,217 1,106 1,133 443 989 1,070 1,196 926 677 479 539 1,283 998 1,148 1,310 545 398 707
No. of differencesc
1
a
“C” denotes an ORF on the negative strand, while the absence of remarks in the strand column denotes the positive strand. NCBI accession number U68299. Differences between MCMV-WT1 and Rawlinsons’s MCMV sequence, as determined by the Crossmatch algorithm in Consed, were enumerated in each predicted ORF (9). They do not precisely correspond to the data shown in Fig. 1, which displays the number of differences in each 100-bp, nonoverlapping window. b c
(Fig. 1 and Table 1). There were 452 differences, including 50 insertion/deletions (indels) and 402 single-bp substitutions. While some of these differences could represent sequencing errors in the original Rawlinson sequence, we visualized the differences between MCMV-WT1 and the Rawlinson sequence in nonoverlapping 100-bp windows across the genome (Fig. 1). Whereas the majority of differences fell into the central coding region, the single window containing the most differences occurred in ORF m04, which encodes an m02 glycoprotein family member. To determine how these differences affected ORFs, we also identified all ORFs previously described (1, 14) (Table 1). Specifically, previously annotated ORFs were located in MCMV-WT1 via a Perl script, which, for each ORF, first searched for an exact match of the entire ORF. If no identical sequence was found, then the script searched for 10-nucleotide sequences which matched the beginning and end of the known
ORF and were separated by the expected distance. If no matches were found, the existing ORFs were aligned to a large region of MCMV-WT1 to find the region of greatest similarity. Because of the large number of indels between MCMV-WT1 and Rawlinson’s annotation, we adjusted the ORF ends for MCMV-WT1 by examining the protein translations of all ORFs and annotating the ends accordingly. Although most ORFs showed comparable protein lengths, the large number of differences between the Rawlinson sequence and our MCMVWT1 made it impossible to attribute any changes in viral function to specific nucleotide changes. Regardless, this high number of differences suggested that MCMV mutated in vivo, as we had previously maintained our MCMV stock by in vivo passages. To elucidate genome stability after in vivo passage, we infected 4-week-old BALB/c mice with MCMV-WT1 and pre-
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TABLE 2. Subpopulation differences in salivary gland stock from BALB/c mice after in vivo passagea No. of shotgun library reads with: Residue
135 190 77068 143110 143111 162252 162328 219363 219365 219366 230248 230287
ORF
None None M52 M98 M98 m107 m108 m159 m159 m159 None None
Difference from consensus
Amino acid change
Mutation
Consensus residue
G3A G3C G3A G3T C3G C3T A3C C3T C3T C3T C3T C3T
NA NA Synonymous Synonymous Leu3Val Arg3Lys Phe3Val Glu3Lys Gly3Lys Gly3Lys NA NA
1 1 1 1 1 2 1 2 2 2 1 1
70 69 31 32 32 35 41 54 54 54 41 41
Mutant fraction (%)
1.4 1.4 3.1 3.0 3.0 5.4 2.4 3.6 3.6 3.6 2.4 2.4
a The corresponding ORF, if previously annotated, is listed, along with the amino acid change, if relevant. To determine the fraction of shotgun clones with the indicated mutation, the number of shotgun clone reads with the indicated mutation are shown along with the corresponding number of shotgun clones with the consensus sequence. The mutant fraction is the number of mutant reads/number of consensus reads ⫻ 100. NA, not applicable.
pared a bulk viral stock from salivary glands 14 days later. Subsequently, we extracted genomic DNA and ligated DNA fragments of this potentially heterogeneous viral stock into shotgun library vectors for sequencing. With a total of 12,642 sequences by the Sanger method, equivalent to 28-fold coverage of the MCMV genome, we found that the bulk salivary stock and the input MCMV-WT1 sequence shared an identical genomic consensus sequence. However, when we used Consed navigator to highlight differences at the individual shotgun library clone level, we identified 12 differences with a Phrap quality value of 40 or greater (Table 2). Several of these differences were identified in both forward and reverse directions, ruling out differences as sequencing errors. These mutations were located randomly throughout the genome, with the possible exception of m159, which contained three mutations, suggesting in vivo selection. There were no mutations in m157, consistent with the absence of Ly49h in BALB/c mice. Excluding the three mutations in m159, the remaining 9 mutations allowed us to estimate the mutation rate of MCMV as ⬃1.0 ⫻ 10⫺7 mutations per bp per day after in vivo passage, very similar to the mutation rate calculated for in vitro passage. To analyze in vivo mutation in a different system, we used B6.BXD8/RAG1KO, a novel murine strain deficient in both adaptive immunity and Ly49h on a C57BL/6 genetic background (3). Since m157 mutations occurred as a result of Ly49H immune selection, we hypothesized that no mutations would occur in m157 in the absence of Ly49H expression. We infected 8-week-old B6.BXD8/RAG1KO mice with MCMVWT1 and harvested spleens 19 to 22 days later. Splenic isolates were plaque purified three times on NIH-3T12 monolayers and then amplified in vitro for 10 to 14 days for genomic DNA extraction. Pyrosequencing reads of each plaque-purified splenic isolate, aligned against the MCMV-WT1 sequence, covered more than 99% of the genomic sequence. Sequence analysis of two independent clones showed that one isolate maintained a sequence identical to MCMV-WT1, while the other isolate differed from MCMV-WT1 at a single residue, a C3A change at residue 52083. This change resulted in a synonymous mutation in predicted ORF m38.5. Moreover, neither splenic isolate demonstrated m157 mutations, contrasting
with our previous finding that 100% of splenic isolates contained mutations in m157 after passage of an MCMV clone with an intact m157 through B6.SCID mice. Furthermore, we PCR amplified an 1,100-bp segment spanning ORF m157 in four other splenic isolates from B6.BXD8/RAG1KO mice. Sequencing of these amplicons revealed only the wild-type sequence (data not shown). Finally, these clones demonstrated no change in virulence compared to that of MCMV-WT1, as measured by splenic viral titers four days postinfection (data not shown). These findings are consistent with our hypothesis that host immune control via Ly49H favored viruses with selective mutations in m157. Moreover, we further confirmed the genomic stability of MCMV after in vivo passage. Here, we examined genomic sequences of Smith strain MCMV and found high genome stability after short-term in vitro and in vivo passages. Whereas previous studies assessed only a small fraction of the genome, we characterized wholegenome sequences via both Sanger method and pyrosequencing. With two independent approaches, we resolved MCMV genomic sequences at the single-nucleotide level. After both in vitro and in vivo passages, we found that Smith strain MCMVWT1 did not acquire functionally significant mutations at a high rate. One caveat to our mutation analysis is that lethal mutations were probably underrepresented in the final DNA pool since, by definition, they did not propagate. Nonetheless, this limitation is intrinsic to all mutation analysis. Lastly, the genomic stability of our MCMV clone could be due to prior, unintentional laboratory selection of MCMV that resulted in a genetically stable virus. Overall, our findings of extremely low mutation rates in vitro and in vivo are consistent with the hypothesis that in the absence of selective pressure, MCMV demonstrates a low mutation rate, comparable to those of other DNA-based microbes (6). Interestingly, previous epidemiologic studies on another herpesvirus family member, human herpesvirus (HSV), delineated ORFs in the Us region as more divergent between HSV-1 and HSV-2, suggesting that mutations preferentially occurred in this area (2). While gene location has been thought to play a role in genome stability (2, 4, 13), in light of our current and previous studies of m157 mutations, the higher
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