Rapid emergence of escape mutants following infection with murine cytomegalovirus in immunodeficient mice

Clinical Immunology 115 (2005) 61 – 69 www.elsevier.com/locate/yclim

Rapid emergence of escape mutants following infection with murine cytomegalovirus in immunodeficient mice Anthony R. Frencha, Jeanette T. Pingelb, Sungjin Kimb, Liping Yangb, Wayne M. Yokoyamab,T a b

Division of Pediatric Rheumatology, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Medicine, Howard Hughes Medical Institute and Division of Rheumatology, Washington University School of Medicine, Box 8045, 660 South Euclid Avenue, St. Louis, MO 63110, USA Received 14 February 2005; accepted 14 February 2005 Available online 23 March 2005

Abstract Natural killer (NK) cells play a crucial role in the initial host defense against pathogens such as murine cytomegalovirus (MCMV). They respond rapidly and effectively control pathogen replication while the adaptive immune system is being activated. However, in the absence of an adaptive immune system, an effective initial NK cell response is not sufficient for long-term pathogen control as demonstrated by the late recrudescence of disease and mortality in immunodeficient mice infected with MCMV. In this setting, NK cells suppress the initial infection but exert enough selective pressure to drive the outgrowth of MCMV mutants that escape recognition by NK cells. Herein, we characterize the rapid emergence of escape mutants following infection with a plaque-purified MCMV isolate and demonstrate that these mutant viruses are no longer effectively controlled by NK cells. These findings suggest that late recrudescence of viral infections in certain clinical settings may also be due to viral escape from NK cells or other components of innate immunity. D 2005 Elsevier Inc. All rights reserved. Keywords: MCMV; m157; Viral escape mutants; NK cells; Innate immunity

Introduction Natural killer (NK) cells are important components of the initial innate immune defense against pathogens and are particularly critical in responding to viral infections (reviewed in [1,2]). Indeed, the hallmark of selective human NK cell deficiencies is difficulty with herpesvirus infections ([3]; reviewed in [4]). In mice, depletion of NK cells results in increased susceptibility to a number of viruses including murine cytomegalovirus (MCMV) [5]. The early defense against pathogens provided by NK cells and other components of the innate immune system is crucial to host survival, since adaptive immunity becomes manifest only after clonal expansion of antigen-specific T and B cells [6,7]. In the absence of an effective innate immune

T Corresponding author. Fax: +1 314 362 9257. E-mail address: [email protected] (W.M. Yokoyama). 1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.02.008

response, pathogen replication may be so rapid that the pathogen overwhelms the host, resulting in death within the first few days of infection [8,9]. NK cell mediated control of MCMV is genetically determined by Cmv1, an autosomal dominant locus encoding the NK cell activation receptor Ly49H [8,10–12]. Strains of mice expressing this NK cell receptor, such as C57BL/6 (B6) mice, are significantly more resistant to MCMV infection than strains of mice that lack Ly49H expression [8,10–13]. Ly49H specifically recognizes the MCMV-encoded open reading frame (ORF) product m157 on virally infected cells and triggers NK cell mediated responses including cytotoxicity and cytokine production [14,15]. Mice with intact adaptive immune systems but genetically lacking Ly49h, such as BALB/c (Cmv1 s allele) mice, can survive infection with low doses of MCMV but are susceptible to infections with higher inoculi of MCMV as evidenced by elevated splenic viral titers and increased mortality [10]. In contrast, Ly49H+ (Cmv1 r ) murine strains,

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such as B6 mice or BALB/c mice reconstituted with Ly49h by transgenesis, have low viral titers and survive infection with similar inoculi of MCMV [8,10–13]. Viruses employ a number of strategies to evade innate and adaptive immune responses (reviewed in [16–19]). Small RNA viruses such as lymphocytic choriomeningitis virus (LCMV), HIV, and hepatitis C replicate rapidly with high mutation rates, resulting in highly heterogeneous viral quasispecies that allow the virus to evade some components of the innate or adaptive immune systems [17,20,21]. For example, RNA viruses can evade the effects of neutralizing antibodies by the rapid generation and outgrowth of monoclonal antibody resistant (MAR) mutants [22–26]. Alternatively, selective pressure imposed by cytotoxic T lymphocytes (CTLs) can lead to CTL escape mutants during infection with simian immunodeficiency virus, HIV, or hepatitis C virus [20,27–30]. In contrast, large DNA viruses have evolved a number of alternative immunoevasion strategies (reviewed in [16–19]) since their lower mutation rates are thought to preclude the generation of escape mutants in a similar fashion to RNA viruses. MCMV is a h-herpesvirus that, like other large double-stranded DNA viruses, has evolved a genome containing multiple ORFs involved in immunoevasion [31,32]. For example, the MCMV genome contains several ORFs that effectively down-regulate MHC class I expression, facilitating evasion from CD8+ CTLs (reviewed in [16,18,33]). MCMV also encodes several ORFs that interfere with innate immune responses including ORFs m152 and m155 which down-regulate ligands for the NK cell activation receptor NKG2D [34,35] and ORF m144 which appears to inhibit NK cells through an as yet undefined inhibitory NK cell receptor [16,36,37]. Despite the lower mutation rates of DNA viruses compared to RNA viruses, the estimated mutation rates of DNA viruses are higher than those of host genes [38–41], leaving open the possibility that, under certain circumstances, DNA viruses may evade host immune responses by generating escape mutants. Indeed, the emergence of heterogeneous antibody resistant escape mutants of the single-stranded DNA minute virus of mice (MVMi) has been reported [42]. In a recent paper, we demonstrated that, in the absence of an adaptive immune response, NK cells exerted enough selective pressure to facilitate the outgrowth of escape mutants of MCMV during the course of a single infection [43]. Therefore, early NK cell mediated control of MCMV infection was inadequate for long-term survival of B6 mice lacking an adaptive immune system. Herein, we extend these observations and further characterize the rapid emergence of MCMV escape mutants that arise after infection with plaque-purified MCMV in mice lacking an adaptive immune response. These studies have significant implications for the clinical care of immunocompromised patients since they suggest that recurrent, severe viral infections in these patients may represent mutants escaping innate immunity.

Materials and methods Mice C57BL/6 (B6), C57BL/6-SCID (B6-SCID), and C57BL/ 6-RAG1 / (B6-RAG) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). B6 mice were also utilized from NCI (Charles River, MA). Congenic B6 mice with a BALB/c haplotype of the NK gene complex (B6.BALB-Cmv1 s ) were a generous gift from A. Scalzo (University of Western Australia). Mice were maintained under specific pathogen-free conditions and used between 8 and 14 weeks of age. All experiments were conducted in accordance with institutional guidelines for animal care and use. Virus and infection of mice Smith strain MCMV was a generous gift from H. Virgin (Washington University, St Louis, MO). A salivary gland stock of MCMV was prepared from BALB/c mice that had been intraperitoneally (i.p.) injected with tissue-culturepropagated MCMV, and the titer was determined via standard plaque assay using permissive NIH 3T12 fibroblasts (American Type Culture Collection, ATCC, Manassas, VA) [8]. For in vivo experiments, mice were injected i.p. with 2  104 to 1  105 plaque-forming units (pfu)/ mouse of a salivary gland MCMV stock. Alternatively, some experiments were performed with tissue-culturepropagated, plaque-purified MCMV (with verified m157 sequence), as indicated. An intentional mutant of m157 and its revertant [44] were generously provided by U. Koszinowski (Max von Pettenkoffer-Institute, Munich, Germany) and S. Jonjic (University of Rijeka, Crotia). Plaque assays Viral titers were determined by standard plaque assay [8]. Briefly, spleens from infected mice were weighed and frozen. Spleens were later thawed and homogenized with Dounce homogenizers on ice. Serial dilutions of the splenic lysates were used to infect monolayers of NIH 3T12 fibroblasts in triplicate on six-well plates. Titers were read by two independent observers on day four using a light microscope with 4 magnification. Titers are presented as log (pfu/100 mg spleen). Propagation of mutant virus Individual viral plaques were isolated from monolayers of NIH 3T12 cells 5 days after inoculation with splenic homogenates of B6-SCID mice harvested at day 28 postinfection (p.i.). The individual viral plaques were expanded by in vitro propagation in monolayers of NIH 3T12. After m157 sequencing, ten of these mutant strains of virus (termed SCID-MCMV isolates) were propagated in MCMV-

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sensitive BALB/c mice to generate salivary gland stocks for in vivo experiments. In addition, tissue-culture-propagated stocks were generated for some experiments. Viral genetic analysis MCMV genomic DNA was extracted using QIAmp DNA Blood minikits (Qiagen). MCMV ORF m157 was PCR amplified using TAQ polymerase (Promega) with the following primers (10 pmol/reaction) CTT GTT AGT GCC GGT GTC TGT and CAT GGT ACA CAA ACG CAG A. PCR products were purified with QIAquick PCR purification kits (Qiagen). Sequencing reactions were prepared using ABI Big Dye Terminators v 2.0 or v 3.1 following standard ABI protocols. Sequencing was performed on a 377 DNA Sequencer (ABI Prism) as well as by the core sequencing facility at Washington University.

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2  104 pfu/mouse of MCMV. A control group of infected B6-SCID mice received an i.v. injection with media alone. Splenocyte preparation, antibody staining, and flow cytometry Three days after MCMV infection, mice were euthanized and single-cell suspensions of splenocytes were prepared using standard techniques [45]. The splenocytes were stained with PerCP-145-2C11 (CD3, Pharmingen), APCPK136 (NK1.1, Pharmingen), PE-streptavidin (Pharmingen), biotinylated-3D10 (Ly49H, [46]), or biotinylated-4E4 (Ly49D, [47]). To block non-specific binding of antibodies to Fc receptors, all antibodies were diluted in the presence of mAb 2.4G2 (Fcg II/III receptor, ATCC). Ly49H+ NK cell populations were then analyzed with flow cytometry. Statistical analysis

Reporter assay with MCMV-infected macrophages The generation and characterization of HD12 reporter cells have been previously described [15]. One day prior to co-culture with the HD12 reporters, IC-21 macrophages (ATCC) were infected with MCMV isolates at a multiplicity of infection (MOI) of 5. Infection of the IC-21 macrophages was verified by characteristic morphological changes in the cells 24 h after infection as well as by flow cytometric analysis following staining with unlabelled antim144 antibodies (ATCC) and PE-goat anti-mouse IgG antibodies (Caltag Lab). In control experiments, HD12 reporters were pre-incubated with 10 Ag/ml of F(abV)2 fragments of anti-Ly49H monoclonal antibodies prior to incubation with infected IC-21 cells [8,15]. HD12 cells were also co-cultured with BaF3 cells transfected either with an empty vector or with m157 as a positive control. The generation of the transfected BaF3 cell lines was previously described [15]. After co-culture of HD12 reporters with infected IC-21 cells, h-galactosidase activity was quantitatively measured with the substrate chlorophenol red h-d-galactoside (CPRG; Calbiochem). h-galactosidase activity in the HD12 reporters was normalized to the maximum h-galactosidase activity induced by phorbol 12-myristate 13-acetate (PMA) (0.5 ng/ml) and ionomycin (1 Ag/ml). Adoptive transfer of immune splenocytes Spleens were harvested from B6.BALB-Cmv1 s mice on day 14 p.i. with 2  104 pfu/mouse of MCMV. Single-cell suspensions of splenocytes were prepared using standard techniques [45], and viable lymphocytes were isolated with a Lympholyte M density separation following the manufacturer’s recommendations (Cedarlane; Ontario, Canada). Immune splenocytes (3.5  106/mouse) were then injected intravenously (i.v.) via retro-orbital vein into a cohort of B6-SCID mice which had been previously infected with

Heteroscedastic two-tailed Student’s t test was used to determine statistically significant differences (P b 0.05). Survival curves Mice were injected i.p. with wt MCMV or MCMV-SCID isolates at the indicated dosages and observed daily for 28 days. Moribund mice were euthanized per institutional guidelines.

Results Late deaths and elevated viral titers observed during MCMV infection in mice lacking an adaptive immune response Immunodeficient mice on a B6 background (B6-SCID or B6-RAG mice) initially control MCMV infection in a manner very similar to wild-type (wt) B6 mice. However, over a period of several weeks, these mice experience a recrudescence of MCMV with deaths occurring 3 to 5 weeks post-infection (p.i.). In our initial study, we documented late deaths in a number of cohorts of B6-RAG and B6-SCID mice infected with doses of MCMV as low as 2  104 pfu, inoculi significantly less than the LD50 of MCMV in B6 mice (2.5  105 pfu) [43]. These observations contrast sharply with the much earlier deaths (days 6–7 p.i.) observed in genetically susceptible mice, such as BALB/c, or in otherwise resistant B6 mice with genetic or induced defects in innate immunity including depletion of NK cells [5,8–10]. Our previous work documented that the late deaths observed in the B6-SCID mice correlated with the reemergence of MCMV as measured by elevated viral titers in the spleen and liver [43]. While wt B6 mice were able to control MCMV infection with undetectable splenic titers at day 28 p.i., surviving B6-SCID mice consistently had higher

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titers at day 28 than at day 3 p.i. (Fig. 1B and [43]). Necropsy investigation of B6-SCID mice late during infection (4 weeks p.i.) demonstrated multi-organ system involvement with severe necrosis of the adrenal medulla, suggesting that death occurred secondary to adrenal insufficiency. The recrudescence of disease and late deaths occurred only in immunodeficient and not wt mice, suggesting that the re-emergence of MCMV was due to the absence of an adaptive immune response. We evaluated the role of adaptive immunity in suppressing the re-emergence of MCMV in immunodeficient mice by transferring splenocytes from MCMV-challenged immunocompetent mice into MCMV-infected B6-SCID mice and measuring late splenic titers (Fig. 1A). To avoid any possibility of transferring Ly49H+ NK cells that could affect the resolution of the acute

Fig. 1. Suppression of the re-emergence of MCMV in B6-SCID mice after adoptive transfer of immune splenocytes. (A) Schematic outline of the adoptive transfer experiment. B6-SCID mice were infected with 2  104 pfu/mouse sg MCMV. At day 7 p.i., one group of infected B6-SCID mice received 3.5  106 splenocytes i.v. from previously infected B6.BALBCmv1 s mice and the second group received a control i.v. injection with media alone. (B) Splenic titers at day 28 p.i. are shown for both groups. Splenic titers in B6 mice at day 28 p.i. with 2  104 pfu/mouse from a separate experiment are shown for comparison. The horizontal line represents the limit of detection of the assay.

infection, we used B6 mice congenic for the BALB/c haplotype of the NK gene complex (B6.BALB-Cmv1 s ) as donors; these mice lack Ly49H expression on their NK cells [48]. The transfer of splenocytes containing immune T and B cells completely suppressed splenic titers on day 28 p.i. while the B6-SCID mice that received control injections had elevated splenic titers (Fig. 1B). Therefore, although Ly49H+ NK cells provided effective early control of MCMV, innate immunity in the absence of an adaptive immune response was insufficient for long-term control of MCMV infection. Mutations in m157 in MCMV isolates from immunodeficient mice Since specific stimulation of NK cells during MCMV infection requires Ly49H recognition of MCMV-encoded m157 [14,15], we hypothesized that mutations in m157 may facilitate the re-emergence of MCMV and escape from innate immune control in B6-SCID mice. To test this hypothesis, we isolated plaque-purified MCMV from spleen and liver homogenates of surviving B6-SCID mice at day 28 p.i. (termed SCID-MCMV) and sequenced m157. In our initial study [43], we reported that mutations in m157 were identified in greater than 95% of the splenic (61/64) and hepatic (19/20) SCID-MCMV isolates from eleven mice in three separate experiments performed using salivary-glandpassaged MCMV (sg MCMV) stocks. In striking contrast, sequencing of two adjacent ORFs that belong to the same m145 family in the MCMV genome as m157 revealed no mutations in m158 (13 isolates) or in m159 (12 isolates) [43] even though all three of these ORFs are dispensable for in vitro viral propagation [49]. While there may be additional mutations in other MCMV ORFs that were not detected in our analysis of these three genes, these results clearly demonstrated that m157 mutants were selected during MCMV infection in B6-SCID mice and were disseminated in the periphery. To investigate whether the m157 mutations occurred de novo during the course of infection or were pre-existing as rare subpopulations in the sg MCMV stock, we repeated the experiment with a plaque-purified wt MCMV clone with an intact m157 sequence. Tissue-culture preparations of this plaque-purified MCMV isolate (tc MCMV) were used to infect B6-SCID mice at two different doses. No mice died in this experiment before being euthanized on day 28 p.i. Elevated splenic titers were observed on day 28 p.i. in all five mice infected with 5  105 pfu tc MCMV/mouse but in only two of the five mice infected with 5  104 pfu tc MCMV/mouse (Fig. 2), supporting the hypothesis that the emergence of m157 mutants is dependent on the initial inoculum dose. This observation could be interpreted to suggest that mutant MCMV was present in the initial stock and that its outgrowth was dose-dependent; however, the tc MCMV was plaque-purified. In addition, the two mice with elevated titers in the low inoculum group had higher splenic

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two different m157 mutations were identified in each of the mice. In contrast to our previous results sequencing isolates from B6-SCID mice infected with sg MCMV [43], the majority of the mice had an independent, dominant m157 mutation not found in the other mice. These results strongly support the hypothesis that MCMV mutated during the course of infection in vivo and was subsequently selected under pressure from NK cells. Characterization of a SCID-MCMV isolate from plaque-purified tc MCMV infected mice

Fig. 2. Splenic titers at day 28 p.i. in two groups of five B6-SCID mice infected with either 5  104 or 5  105 pfu/mouse of plaque-purified, tissue-culture-propagated MCMV with an intact m157 sequence. The horizontal line represents the level of detection of the assay.

titers than four of the five mice in the higher inoculum group (Fig. 1B), making the argument that the emergence of m157 mutants in these mice simply reflected the outgrowth in a dose-dependent manner of pre-existing, rare subpopulations of m157 mutants substantially less plausible. Three of the five mice in the low inoculum group did not have detectable titers at day 28 p.i., and several mice in the higher inoculum group had lower splenic titers suggesting that the emergence, selection, and accumulation of mutated MCMV may be dependent on the time elapsed post-infection. The strongest evidence that mutations in m157 were occurring de novo during the course of infection came from the sequencing of m157 in 35 splenic isolates from six of the B6-SCID mice infected with plaque-purified tc MCMV (Table 1 where D1–D4 were previously reported in [43]). All the isolates were found to have mutations in m157. Substitutions or deletions resulting in premature stop codons in m157 were identified in 32 of 35 isolates. One or at most

We further characterized one of the SCID-MCMV isolates (D5.3) from the experiment with plaque-purified tc MCMV. Our laboratory had previously generated a Ly49H reporter cell line (HD12) by co-transfecting Ly49H and its signaling partner, KARAP/DAP12, into a BWZ cell line containing a stably integrated h-galactosidase construct that is activated upon ITAM signaling [15]. Ly49H recognition of m157 on infected or transfected cells was identified in the HD12 reporters by a colorimetric change following h-galactosidase induction [15]. Although a macrophage cell line (IC-21) infected with wt MCMV strongly stimulated the HD12 reporter cells, IC-21 cells infected with isolate D5.3 (Fig. 3A) as well as several other SCID-MCMV isolates (generated from mice infected with sg MCMV; Fig. 3B) did not a stimulate the HD12 reporter cells. The baseline hgalactosidase induction stimulated by macrophages infected with the SCID-MCMV isolates was identical to that induced by mock-infected macrophages or macrophages infected with a deliberate m157 mutant, as well as to that induced when the HD12 reporter cells were pretreated with F(abV)2 fragments of anti-Ly49H monoclonal antibodies prior to incubation with macrophages infected with wt MCMV (Fig. 3B). These results demonstrate that recognition of m157 by Ly49H is abrogated following infection with the SCID-MCMV isolates, including isolate D5.3, and complement earlier studies showing that macrophages infected with SCID-

Table 1 Mutations in m157 in splenic SCID-MCMV isolates from B6-SCID mice infected with plaque-purified, tissue-culture-propagated MCMV Mouse

Mutation

Frequency

D1

Insertion of an extra A at 299–304 G644A G538T T179G T840A Deletion of an A between 299 and 304 Insertion of an extra A at 299–304 Deletion of a T at 125

5/7 2/7 8/8 1/8 7/8 5/5 4/4 3/3

D2 D3 D4 D5 D6

Frameshift with stop at 315–317 (TAA) TGT (cys) Z TAT (tyr) Premature stop (TAA) ATT (ile) Z ATG (met) Premature stop (TAA) Premature stop at 311 (TAA) Frameshift with stop at 315–317 (TAA) Frameshift with stop at 168–170 (TAA)

Smith strain MCMV was plaque-purified and sequenced to verify that m157 was intact. After only tissue culture amplification, this MCMV stock was used to infect B6-SCID mice (D1 and D2 at 5  104 pfu/mouse and D3, D4, D5, and D6 at 5  105 pfu/mouse). Homogenates of spleens harvested on day 28 p.i. were used to infect monolayers of NIH 3T12 cells. Five days later, viral plaques were isolated. From each isolate, viral genomic DNA was harvested, and the m157 ORF was PCR amplified for sequencing.

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Fig. 3. Disruption of the Ly49H–m157 interaction during infection with SCID-MCMV isolates. (A) Ly49H recognition of m157 on infected macrophages as assessed by HD12 reporter cells. Reporter cells were co-cultured in triplicate with either mock-infected IC-21 macrophages, IC-21 cells previously infected for 24 h with wt MCMV or SCID-MCMV isolate D5.3, or BaF3 cells transfected with either m157 or an empty vector. Infection of the IC-21 macrophages was verified by staining for m144 expression. (B) Failure of HD12 reporter cells to recognize a panel of IC-21 cells infected with SCID-MCMV isolates A1.04, A1.05, and A1.10. Results are also shown of HD12 reporters pretreated with F(abV)2 fragments of anti-Ly49H monoclonal antibodies prior to incubation with infected IC-21 cells and of HD12 reporters co-cultured with IC-21 cells infected with Dm157 MCMV (deliberate m157 mutation) or its revertant. (C) Absence of selective expansion of splenic Ly49H+ NK cells during infection with SCID-MCMV isolate D5.3. Splenic NK cells were harvested from B6 mice on day 3 p.i. with 5  104 pfu/mouse of wt MCMV or SCID-MCMV isolate D5.3. The percentage of splenic NK cells (NK1.1+ CD3 ) that were Ly49H+ was determined by flow cytometric analysis. The differences between the percentage of Ly49H+ NK cells in naRve B6 mice and mice d3 p.i. with wt MCMV were statistically significant ( P b 0.05) while the differences between naRve B6 mice and mice infected with the SCID-MCMV isolate D5.3 were not. Data represent the averages of groups of four mice and are representative of two experiments. (D) Survival curves of groups of 6 wt B6 mice infected i.p. with 1  105 pfu/ mouse of wt MCMV (open squares) or SCID-MCMV isolate D5.3 (filled triangles).

MCMV isolates fail to induce selective in vitro stimulation of interferon-g in Ly49H+ NK cells [43]. These in vitro observations correlated well with the lack of expansion of splenic Ly49H+ NK cells observed in vivo following infection with SCID-MCMV isolate D5.3 (Fig. 3C) and other SCID-MCMV isolates [43]. This contrasts with the striking accumulation of Ly49H+ NK cells during infection with wt MCMV (Fig. 3C and [43]) reflecting selective Ly49H stimulation and subsequent proliferation of Ly49H+ NK cells [45]. We previously demonstrated that the in vivo defect in specific Ly49H+ NK cell responses illustrated by the absence of selective expansion following infection with SCID-MCMV isolates contrasted with relatively normal early (36 h p.i.) Ly49H-independent bnon-specificQ in vivo stimulation of NK cells (proliferation and IFN-g production) during infection with SCID-MCMV isolates [43]. Therefore, the SCID-MCMV isolates, includ-

ing isolate D5.3, manifest selective abnormalities in the specific in vitro and in vivo recognition by and stimulation of Ly49H+ NK cells. Infection with the SCID-MCMV isolate D5.3 resulted in marked mortality in wt B6 mice by day 6 p.i. compared to 100% survival at 28 days p.i. following infection with wt MCMV, indicating ineffective early innate control of infection with SCID-MCMV isolate D5.3 (Fig. 3D). Similar results (70–100% mortality) have been observed following infection with nine other SCID-MCMV isolates (including 3 shown in [43]) at doses of 6  104 to 1  105 pfu/mouse in 4 independent experiments (data not shown). The LD50 of SCID-MCMV isolate D5.3 was 6  104 pfu, approximately one-fifth the LD50 of wt MCMV, passaged an equivalent number of times in vivo (data not shown). These results demonstrate that the phenotype of uncontrolled viral replication and death observed late during infection with

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sg MCMV in immunodeficient mice could be recapitulated with the SCID-MCMV isolate D5.3 as well as other SCIDMCMV isolates early in the course of infection in naive wt B6 mice.

Discussion The influence of selective pressure by the immune system on viral pathogens has been demonstrated by the mutation of antigenic epitopes for virus-specific immunoglobulins or TCRs [42,50]. These viral escape mutants have been described primarily for RNA viruses due to the inherent low fidelity of RNA polymerase resulting in the propensity of RNA viruses to develop bquasispeciesQ [51]. In almost all cases reported thus far, the viruses escaped adaptive, not innate, immune control. However, we recently demonstrated that a mono-specific innate immune response results in readily detectable mutations in MCMV, a doublestranded DNA virus [43]. In the absence of an adaptive immune response, Ly49H+ NK cells exerted enough selective pressure to drive the specific outgrowth of MCMV escape mutants with alterations in m157 [43]. These SCIDMCMV isolates with m157 mutations, including isolate D5.3, have escaped from Ly49H+ NK cell recognition and abrogated the normal effective control of MCMV in resistant, naRve B6 mice, rendering them as susceptible to MCMV as NK cell depleted or Ly49H-deficient mice [5,8,10]. The striking increased frequency of mutations in m157 in SCID-MCMV isolates compared with the absence of mutations in two adjacent m145 family members (m158 and m159) illustrated that m157 mutants were selected during MCMV infection in immunocompromised mice (Table 1 and [43]). Our initial experiments identified a number of identical m157 mutations in different B6-SCID mice following infection with an in vivo passaged sg MCMV stock, raising the possibility that these mutants may have been present at very low frequencies in the heterogeneous sg MCMV stock. We did not find m157 mutations in individual isolates of the original sg MCMV stock [43]; however, this did not rule out the possible existence of rare subpopulations of m157 mutants, and no conclusion could be drawn regarding whether the mutations occurred during the course of infection or represented the outgrowth of previously existing viral subpopulations. To address this issue, we infected immunocompromised mice with plaque-purified, tc MCMV with a known intact m157 ORF [43]. Sequencing of SCID-MCMV isolates from these mice revealed distinct, independent m157 mutations in each mouse that were different from the mutations identified in the original experiments with sg MCMV (Table 1 and [43]). Therefore, several of the commonly identified mutants in the original experiments may have existed as rare subpopulations in sg MCMV stock that were selected during the course of infection. The identification of distinct,

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dominant mutations in four of the six mice infected with tc MCMV which were not found in other mice within the experiment supported the hypothesis that MCMV mutated during the course of infection in these mice. SCID-MCMV isolates from two of the six mice had an additional adenosine inserted into a stretch of six adenosines (nucleotides 299–304). Interestingly, several other mutations have been observed in this region including a deletion of one of the six adenosines (mouse D4, Table 1) and an 18 nucleotide deletion beginning at nucleotide 305 (A1; [43]). Although the isolation of SCID-MCMV mutants with the addition of an adenosine in this stretch of six adenosines in two mice could suggest a contamination in our tc MCMV stock, the identification of a number of mutations in this area suggests that this region may have a higher mutation rate than the remainder of the m157 ORF. The emergence of m157 mutants in B6-SCID mice infected with plaque-purified tc MCMV occurred relatively rapidly with elevated titers observed on day 28 p.i. in seven of the ten mice. Interestingly, all the mice infected with the higher inoculum of tc MCMV had elevated titers while only two of the five mice infected with the lower inoculum had detectable splenic titers by day 28 p.i., suggesting that a higher inoculum increased the probability of a m157 mutation occurring during this time course. The splenic titers in three of the ten mice (including two in the low inoculum group) were as high as had been observed previously at day 28 p.i. in surviving B6-SCID mice infected with sg MCMV (Fig. 1B and [43]), suggesting that mutations may occur fairly rapidly, allowing substantial dissemination and accumulation of mutated virus. However, the variability in splenic titers within the high inoculum group implicated some stochastic component in the acquisition and accumulation of mutated virus. Taken together, the distribution of elevated splenic titers between the two groups, the variation in absolute splenic titers within the two groups, as well as the presence of distinct, independent mutations in individual B6-SCID mice following infection with tc MCMV demonstrate that the emergence of m157 mutants is dependent on the initial inoculum dose of MCMV as well as length of time post-infection and supports the hypothesis that m157 mutations occur de novo and relatively rapidly during the course of infection. We did not detect mutations in m157 during the course of infection in immunocompetent mice, presumably due to effective suppression by CTLs and other adaptive immune components providing a multi-pronged response to MCMV. Indeed, the adoptive transfer of immune T and B cells into B6-SCID mice 1 week after infection with MCMV completely suppressed the emergence of m157 mutations. However, recent studies have identified m157 mutations after multiple rounds of sequential salivary gland passage of K181 strain MCMV in immunocompetent congenic Ly49H+ mice [52]. It remains to be determined how widely distributed mutations in m157 are in outbred wild mice; however, mutations in m157 have been identified in MCMV

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isolates from wild mice [52]. Although the function of m157 in vivo is not well characterized, it presumably impacts viral virulence beyond being a ligand for Ly49H, and mutations in m157 may be detrimental to other aspects of modulating host immune responses. Further study is needed to address these issues. We believe that the rapid emergence of escape viruses in immunocompromised mice has significant implications for the clinical care of immunocompromised patients. The absence of an effective adaptive immune response in immunodeficient patients (such as SCID patients, AIDS patients, or immunosuppressed organ transplant patients) frequently results in severe viral illnesses, such as CMV viremia, meningitis, pneumonia, and retinitis, similar to the widespread disease in our infected B6-SCID mice [53–55]. Interestingly, immunosuppressed transplant recipients who receive a lung transplant from a CMV-positive donor typically experience CMV disease relatively late (more than a month) rather than acutely after the transplant [54]. This time course is reminiscent of our studies in which the reemergence of MCMV occurred several weeks after control of the initial infection due to the selection and subsequent outgrowth of m157 mutants. We, therefore, hypothesize that certain viral infections in immunodeficient patients may become clinically evident when viruses escape from innate immune control and that genomic analysis of such clinical isolates may provide valuable insight into the viral products that interact with the host innate immune system.

Acknowledgments This work was supported by a KO8 grant (AI059083) from the NIAID and a HHMI Faculty Development Award to ARF, and by the Barnes-Jewish Hospital Research Foundation and NIH grants to WMY who is a HHMI investigator. The authors gratefully acknowledge critical reading of this manuscript by Joy Loh and Randall Rodrigues.

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