Stability of Murine Cytomegalovirus Genome after In Vitro and In Vivo Passage

Abstract

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.

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 Cmv1^r allele, which encodes the Ly49H activation receptor on NK cells (7, 19). The only known ligand for Ly49H is MCMV-encoded 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 Cmv1^r 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 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 MCMV-WT1, -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 G→A change at residue 8847 and an A→G 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) (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 MCMV-WT1 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.

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.

TABLE 1.

Predicted ORFs in MCMV-WT1 compared to those in the previously published sequence

ORF	Stranda	MCMV-WT1			Rawlinson's MCMV sequenceb			No. of differencesc
		Start	End	ORF length	Start	End	ORF length
m01	C	468	870	402	480	836	356	1
m02		1033	2013	980	999	1979	980
m03		2270	3109	839	2236	3102	866	15
m04		3267	4063	796	3270	4070	800	74
m05		4179	5200	1,021	4185	5210	1,025	31
m06		5291	6336	1,045	5300	6337	1,037	3
m07		6463	7407	944	6463	7407	944
m08		7459	8529	1,070	7459	8529	1,070
m09		8632	9513	881	8632	9513	881
m10		9624	10499	875	9624	10499	875
m11		10715	11614	899	10715	11614	899
m12		11686	12504	818	11686	12504	818
m13		12599	13000	401	12599	13000	401
m14		13085	13990	905	13085	13990	905
m15		14085	15065	980	14085	15065	980
m16		15044	15676	632	15044	15676	632
m17	C	15749	16951	1,202	15749	16951	1,202
m18	C	17071	20193	3,122	17071	20193	3,122
m19		20338	20781	443	20338	20781	443
m20	C	20579	23044	2,465	20802	23045	2,243	1
m21		22644	23333	689	22645	23334	689
m22		23585	23899	314	23586	23900	314
M23	C	23777	24952	1,175	23778	24953	1,175
m23.1		24825	25160	335	24826	25161	335
M24	C	25147	26118	971	25148	26119	971
M25		26014	28812	2,798	26015	28813	2,798
m25.1	C	28997	30601	1,604	28998	30602	1,604
m25.2	C	28997	30280	1,283	28998	30281	1,283
m25.3	C	30244	31656	1,412	30245	31657	1,412
m25.4	C	30244	31215	971	30245	31216	971
M26	C	31346	31924	578	31347	31925	578
M27	C	32247	34295	2,048	32247	34295	2,048
M28	C	34486	35778	1,292	34486	35778	1,292
m29		35747	36475	728	35747	36730	983	1
m29.1	C	36030	36661	631	36109	36660	551	1
m30		36885	39071	2,186	36884	37729	845	1
M31		37281	39071	1,790	37279	38829	1,550	1
M31b		38777	39079	302	38775	39065	290	1
M32	C	39283	41439	2,156	39280	41436	2,156
M34		43086	45650	2,564	43083	45647	2,564
M35		45912	47471	1,559	45909	47468	1,559
M37	C	49444	50481	1,037	49441	50478	1,037
M38	C	50465	51958	1,493	50462	51955	1,493
m38.5c	C	51783	52523	740	51780	52520	740
m39	C	52487	53203	716	52484	53200	716
m40	C	53268	53633	365	53265	53630	365
m41	C	53786	54202	416	53783	54199	416
m42	C	54355	54846	491	54352	54843	491
M43	C	55354	57147	1,793	55351	57144	1,793
M44	C	57888	59123	1,235	57885	59120	1,235
m44.1		58759	60108	1,349	58756	60105	1,349
m44.3		59144	59428	284	59141	59425	284
M45e1	C	59518	62160	2,642	59515	62145	2,630	1
M45e2	C	62773	62880	107	62769	62876	107
m45.1	C	59520	63042	3,522	61764	63038	1,274	1
m45.2		62810	62890	80	62806	62886	80
M46	C	63044	63928	884	63040	63924	884
m48.1		73566	73877	311	73562	73873	311
m48.2	C	73575	73871	296	73571	73867	296
M49	C	73923	75533	1,610	73919	75529	1,610	1
M50	C	75505	76455	950	75501	76451	950	5
M51	C	76519	77220	701	76515	77216	701	1
M52		76919	78471	1,552	76915	78468	1,553	2
M53		78465	79462	997	78461	79462	1,001	3
M55	C	83004	85811	2,807	83003	85816	2,813	61
M56	C	85711	88107	2,396	85716	88112	2,396	1
m58		91756	92459	703	91761	92465	704	8
m59		93236	94393	1,157	93241	94263	1,022	5
M69	C	96284	98812	2,528	96193	98721	2,528
m69.1		98621	98979	358	98530	98889	359	1
M70	C	99101	101995	2,894	99010	101904	2,894	2
M71		101994	102893	899	101903	102802	899
M72	C	103122	104327	1,205	103031	104236	1,205	5
M73		104191	104609	418	104100	104519	419	8
M73.5e2		105888	106160	272	105797	106069	272
m74	C	104587	105903	1,316	104496	105812	1,316	19
M75	C	106205	108382	2,177	106110	108287	2,177	30
M76		108479	109242	763	108384	109148	764	2
M77		109026	110912	1,886	108931	110817	1,886
M78		111084	112498	1,414	110989	112404	1,415	4
M79	C	112737	113513	776	112639	113415	776	2
M80		113512	115607	2,095	113414	115507	2,093	11
M82	C	115812	117611	1,799	115711	117507	1,796	6
M83	C	117718	120147	2,429	117614	120043	2,429	13
M84	C	120186	121949	1,763	120082	121845	1,763	10
M85	C	122293	123228	935	122189	123124	935	1
M87		127487	130267	2,780	127383	130163	2,780
M88		130347	131626	1,279	130243	131523	1,280	2
m90	C	133020	133976	956	132920	133876	956
M91		133768	134172	404	133668	134072	404
M92		134175	134867	692	134075	134767	692
M93		134833	136379	1,546	134733	136280	1,547	1
M94		136334	137370	1,036	136234	137271	1,037	11
M89Ex1	C	137487	138380	893	137390	138283	893	1
M95		138379	139632	1,253	138282	139535	1,253
M96		139632	140021	389	139535	139924	389
M97		140238	142168	1,930	140141	142072	1,931	1
M98		142198	143883	1,685	142101	143786	1,685
M99		143820	144158	338	143723	144061	338
M100	C	144393	145508	1,115	144296	145411	1,115
M102		145693	148131	2,438	145596	148034	2,438
M103	C	148279	149232	953	148182	149135	953
M104	C	149210	151324	2,114	149113	151227	2,114
M105		151125	153971	2,846	151028	153874	2,846
m106	C	154010	154453	443	153913	154356	443
m106.1	C	154293	154553	260	154196	154456	260
m106.3	C	155878	156015	137	155781	155918	137
m107		162083	162777	694	161983	162678	695	2
m108	C	162310	162870	560	162210	162770	560
M114	C	165696	166484	788	165596	166384	788
M115	C	166484	167308	824	166384	167208	824
M116	C	167305	169242	1,937	167205	169142	1,937
m117	C	169313	171010	1,697	169213	170910	1,697
m117.1		169641	171055	1,414	169541	170956	1,415	1
M118	C	171080	172045	965	170980	171945	965
m119.1	C	172156	173091	935	172056	172991	935
m119.2	C	173122	173490	368	173022	173390	368
m119.3	C	173510	173821	311	173410	173721	311
m119.4	C	174154	174435	281	174054	174335	281
m119.5		174254	174589	335	174154	174489	335
m120	C	174399	174674	275	174299	174574	275
m120.1	C	174740	175825	1,085	174640	175725	1,085
M121	C	175779	177875	2,096	175679	177775	2,096
M121	C	175779	177875	2,096	175679	177775	2,096
m123.1	C	181963	182319	356	181863	182219	356
m123Ex2	C	181756	181866	110	181656	181766	110
m124		182033	182380	347	181933	182280	347
m124.1	C	182111	182518	407	182011	182418	407
m125		183536	183865	329	183436	183765	329
m126		184635	184910	275	184535	184810	275
m127	C	185290	185691	401	185190	185591	401
m128Ex3		186185	187399	1,214	186085	187299	1,214
m129	C	187447	187947	500	187347	187847	500
m130		187907	188380	473	187807	188280	473
m131	C	188126	188476	350	188026	188376	350
m133Ex1	C	188978	189895	917	188878	189795	917
m134		189968	190381	413	189868	190281	413
m135	C	189995	190321	326	189895	190221	326
m136	C	190410	191171	761	190310	191071	761
m137	C	191188	192192	1,004	191088	192092	1,004
m138	C	192333	194042	1,709	192233	193942	1,709
m139	C	194182	196116	1,934	194082	196016	1,934
m140	C	196162	197616	1,454	196062	197516	1,454
m141	C	197805	199331	1,526	197705	199231	1,526
m142	C	199541	200848	1,307	199441	200748	1,307
m143	C	201065	202694	1,629	200920	202593	1,673	1
m144	C	202843	203994	1,151	202742	203893	1,151
m145	C	204130	205593	1,463	204029	205492	1,463
m146	C	205743	206876	1,133	205642	206775	1,133
m147	C	206963	207400	437	206862	207299	437
m148		207029	207388	359	206928	207287	359
m149		207427	208116	689	207326	208015	689
m150	C	207724	208890	1,166	207623	208789	1,166
m151	C	208915	210084	1,169	208814	209983	1,169
m152	C	210342	211478	1,136	210241	211377	1,136
m153	C	211688	212905	1,217	211587	212804	1,217
m154	C	213043	214149	1,106	212942	214048	1,106
m155	C	214535	215668	1,133	214434	215567	1,133
m156	C	215635	216078	443	215534	215977	443
m157	C	215996	216985	989	215895	216884	989
m158	C	217033	218103	1,070	216932	218002	1,070
m159	C	218271	219467	1,196	218170	219366	1,196
m160	C	219699	220625	926	219598	220524	926
m161	C	220573	221250	677	220472	221149	677
m162	C	221287	221766	479	221186	221665	479
m163	C	221976	222515	539	221875	222414	539
m164	C	222467	223750	1,283	222366	223649	1,283
m165	C	223381	224379	998	223280	224278	998
m166	C	224514	225662	1,148	224413	225561	1,148
m167	C	225880	227190	1,310	225779	227089	1,310
m168		228021	228566	545	227920	228465	545
m169	C	228411	228809	398	228310	228708	398
m170	C	229440	230147	707	229339	230046	707

^a“C” denotes an ORF on the negative strand, while the absence of remarks in the strand column denotes the positive strand.

^bNCBI accession number U68299.

^cDifferences 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.

To elucidate genome stability after in vivo passage, we infected 4-week-old BALB/c mice with MCMV-WT1 and prepared 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.

TABLE 2.

Subpopulation differences in salivary gland stock from BALB/c mice after in vivo passage^a

Residue	ORF	Difference from consensus	Amino acid change	No. of shotgun library reads with:		Mutant fraction (%)
				Mutation	Consensus residue
135	None	G→A	NA	1	70	1.4
190	None	G→C	NA	1	69	1.4
77068	M52	G→A	Synonymous	1	31	3.1
143110	M98	G→T	Synonymous	1	32	3.0
143111	M98	C→G	Leu→Val	1	32	3.0
162252	m107	C→T	Arg→Lys	2	35	5.4
162328	m108	A→C	Phe→Val	1	41	2.4
219363	m159	C→T	Glu→Lys	2	54	3.6
219365	m159	C→T	Gly→Lys	2	54	3.6
219366	m159	C→T	Gly→Lys	2	54	3.6
230248	None	C→T	NA	1	41	2.4
230287	None	C→T	NA	1	41	2.4

^aThe 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.

To analyze in vivo mutation in a different system, we used B6.BXD8/RAG1^KO, 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/RAG1^KO mice with MCMV-WT1 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 C→A 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/RAG1^KO 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 whole-genome 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 MCMV-WT1 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 gene divergence may reflect differences in viral pathogenesis. Specifically, in our current study, MCMV did not appear to preferentially accumulate mutations in specific loci (except for, possibly, m159) in the absence of obvious selective pressure. In other words, an alternative hypothesis for the finding of mutations concentrating in certain loci is that they reflect host selective pressure, as seen in the setting of B6 mice which possess Cmv1^r (Ly49h).

Nucleotide sequence accession number.

The GenBank accession number for MCMV-WT1 is GU305914.