JOURNAL OF VIROLOGY, Oct. 2009, p. 10293–10298
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 19
Immune Evasion Proteins Enhance Cytomegalovirus Latency
in the Lungs?
Verena Bo ¨hm, Christof K. Seckert, Christian O. Simon, Doris Thomas, Ange ´lique Renzaho,
Dorothea Gendig, Rafaela Holtappels, and Matthias J. Reddehase*
Institute for Virology, University Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany
Received 4 June 2009/Accepted 7 July 2009
CD8 T cells control cytomegalovirus (CMV) infection in bone marrow transplantation recipients and persist
in latently infected lungs as effector memory cells for continuous sensing of reactivated viral gene expression.
Here we have addressed the question of whether viral immunoevasins, glycoproteins that specifically interfere
with antigen presentation to CD8 T cells, have an impact on viral latency in the murine model. The data show
that deletion of immunoevasin genes in murine CMV accelerates the clearance of productive infection during
hematopoietic reconstitution and leads to a reduced latent viral genome load, reduced latency-associated viral
transcription, and a lower incidence of recurrence in lung explants.
Establishment of latency after clearance of acute infection
and the potential to reactivate to recurrent infection are key
features of herpesvirus pathogenicity (41). Cytomegaloviruses
(CMVs) are usually well controlled by the immune system and
cause acute as well as recurrent disease mainly in the immu-
nocompromised or immunologically immature host. Recipi-
ents (R) of bone marrow transplantation (BMT) are at risk of
reactivating intrinsic human CMV (hCMV) or of becoming
infected by reactivating donor (D)-derived hCMV transmitted
with the transplant or of both (D?R?, D?R?, and D?R?
constellations, respectively) (9). Clinical studies (32, 38) and
experimental studies of the murine model of infection with
murine CMV (mCMV) (18; reviewed in reference 16) have
consistently shown that timely endogenous lymphohematopoi-
etic reconstitution of antiviral CD8 T cells is decisive for cop-
ing with CMV infection after BMT. Accordingly, preemptive
immunotherapy with antiviral CD8 T cells proved to be a
promising approach with the murine model (3, 14, 35, 36, 44)
and in clinical trials (6, 27, 40).
Both viruses hCMV and mCMV express proteins, so-called
immunoevasins, that interfere with the major histocompatibil-
ity complex class I pathway of antigen presentation to CD8 T
cells (reviewed in reference 33). Whereas many studies have
demonstrated the efficacy of immunoevasins in inhibiting the
cell surface presentation of antigenic peptides in infected cells
in vitro, these molecules do not prevent (11, 19, 26) but rather
enhance (4) the priming of viral epitope-specific CD8 T cells,
and their role and relevance in viral pathogenesis in vivo are a
currently discussed issue (8). Obviously, research on the in vivo
function of immunoevasins by using viral immunoevasin gene
deletion mutants can be accomplished only using animal mod-
els, and the murine model is well established. Although the
detailed molecular modes of action differ between the immu-
noevasins of hCMV and mCMV, the biological outcome in
* Corresponding author. Mailing address: Institute for Virology, Uni-
versity Medical Center of the Johannes Gutenberg University, Hochhaus
am Augustusplatz, 55131 Mainz, Germany. Phone: 49-6131-39-33651.
Fax: 49-6131-39-35604. E-mail: Matthias.Reddehase@uni-mainz.de.
?Published ahead of print on 15 July 2009.
FIG. 1. Scheme illustrating the strategy for studying viral latency in the
lungs. (A) Protocol of BMT. Syngeneic BMT was performed with 8- to
10-week-old female BALB/c mice as donors and recipients of bone marrow
cells (BMC) as described in greater detail previously (29). Briefly, recipients
were immunocompromised by total-body ? irradiation with a single dose of
6.5 Gy at 6 h prior to the intravenous transfer of 5 ? 106pooled femoral and
tibial BMC and were infected with 105PFU of either mCMV-WT.BAC or
mCMV-?vRAP in the left hind footpad at 2 h after BMT. The time course
of infection of the lungs was monitored until latency was established.
(B) Analysis of latency and reactivation. Shown is the lobular anatomy of the
mouse lungs in ventral view. After the lungs were cut into 18 pieces, each
representing ?3 million cells, lung pieces 1 through 7, derived from the left
lung, were used for tissue explant cultures to determine the cumulative inci-
18, derived from the inferior, middle, and superior lobes of the right lung,
were used for the quantitation of IE1 transcripts.
both instances is the inhibition of antigen presentation. Thus,
there is good reason to assume that the murine model also
gives us valuable predictions for the in vivo role of hCMV
Three molecules that regulate antigen presentation to CD8
T cells are known for mCMV. The immunoevasins m152/gp40
(7, 47) and m06/gp48 (37) interfere with the vesicular transport
of peptide-loaded major histocompatibility complex class I
molecules. Although m04/gp34 may cooperate with these two
confirmed immunoevasins, more-recent data with a mutant
virus expressing m04/gp34 selectively have revealed that it is no
CD8 T-cell immunoevasin in its own right (15, 28). A virus
lacking all three “viral regulators of antigen presentation”
(vRAPs), the deletion mutant mCMV-?m04?m06?m152
(45), here referred to as mCMV-?vRAP, is used to study the
immune response and viral pathogenesis in the absence of
vRAPs. Importantly, a previous study has shown that deletion
of the vRAP genes does not affect viral replicative fitness in
immunocompromised mice, as demonstrated by unaltered
doubling times in various host tissues (4) compared with results
for bacterial artificial chromosome (BAC)-cloned wild-type
(WT) virus (46), mCMV-WT.BAC. Therefore, any in vivo
growth phenotype of mutant virus mCMV-?vRAP in immu-
nocompetent mice or during immunological reconstitution af-
ter BMT can be attributed to immunological control.
Previous studies of immunocompetent C57BL/6 and B-cell-
deficient ?MT mice, both of which are resistant to mCMV due
to natural killer (NK) cell activation (1), have suggested that
vRAPs have little impact on virus replication, establishment of
latency, and virus reactivation upon immunosuppression (12),
with the exception of elevated virus titers in salivary glands of
mCMV-susceptible BALB/c mice (25). As introduced above, it
is a hallmark of CMV biology that infection with WT CMVs is
well controlled by the immune system despite the expression of
immunoevasins, so in immunocompetent mice, only incremen-
tal improvement can be expected from the deletion of immu-
noevasin genes. An impact of vRAPs might rather be seen in
the immunocompromised host, especially in the clinically rel-
evant situation of lymphohematopoietic reconstitution of an-
tiviral CD8 T cells in BMT recipients. Importantly, whereas
CD8 T cells can be replaced with other innate and adaptive
effector cells in otherwise immunocompetent mice (20), anti-
viral CD8 T cells are essential for preventing CMV disease in
the BMT setting (30, 31). Although deletion of vRAP m152/
gp40 can also activate NK cells through expression of the
activating NKG2D ligand RAE-1 (for a review, see reference
24), previous work has demonstrated that CD8 T cells outper-
form NK cells in controlling the in vivo replication of mCMV-
?vRAP (4). Since CD8 T cells and NK cells are regulated in
parallel by m152/gp40, both may contribute to latency in the
same direction. Altogether, experimental BMT in the mouse
should be a good model for unraveling a potential in vivo role
Here we focused on infection of the lungs, since interstitial
pneumonia is a very relevant manifestation of CMV disease in
BMT recipients (39) and since the lungs are a major organ site
of mCMV latency after neonatal infection (2) and after exper-
imental BMT (22, 30). Figure 1 sketches the experimental
protocol of syngeneic BMT (Fig. 1A) and the analysis of viral
latency (Fig. 1B). As shown in Fig. 2A by the time course of
viral replication in the recipients’ lungs, mutant virus repli-
cated like WT virus only in the first 2 weeks, which is consistent
with unaltered replicative fitness in the absence of immune
cells (4). In contrast, significantly more-efficient control of the
FIG. 2. Pulmonary infection and immune response after BMT. (A) Time course of viral replication in the lungs of BMT recipients in the
presence (gray circles; WT.BAC) or absence (open circles; ?vRAP) of immunoevasins. Syngeneic BMT (see Fig. 1A), intraplantar infection of the
recipients, and determination of virus titers in lung homogenates by virus plaque (PFU) assay were performed as described previously (see
reference 29 and references therein). Symbols represent data for individual mice. The median values are marked by short horizontal bars. The
statistical significance of differences in virus titers was evaluated by using distribution-free Wilcoxon-Mann-Whitney (rank sum) statistics. Virus
titers in two experimental groups differ significantly if the P value (two-tailed test) is ?0.05. Calculated P values are indicated for group
comparisons of interest. ?, significant difference. DL, detection limit of the assay. (B) Frequencies of epitope-specific CD8 T cells in pulmonary
infiltrates of latently infected mice. Amino acid sequences of the indicated H-2d-restricted antigenic peptides are listed in reference 14. At 6 months
after BMT and infection with WT.BAC (gray bars) or ?vRAP (open bars), lung infiltrate leukocytes were isolated (18, 29) and CD8 T cells were
enriched immunomagnetically (17). Frequencies of gamma interferon-secreting, peptide-sensitized cells were determined by using an enzyme-
linked immunospot assay (see reference 4 and references therein). Bars represent most probable numbers calculated by intercept-free linear
regression analysis. Error bars indicate the 95% confidence intervals. Ø, no peptide added.
10294 NOTESJ. VIROL.
mutant virus was found for all later time points, which corre-
lates with the reappearance of virus-specific CD8 T cells in the
BMT model (18). As shown previously, selective depletion of
CD8 T cells but not of CD4 T cells during lymphohematopoi-
etic reconstitution leads to fatal multiple-organ CMV disease
(31), including a fulminant, disseminated viral interstitial
pneumonia (30). Beyond 16 weeks, productive infection was
resolved for both viruses. Thus, immunoevasins contributed to
higher virus peak levels in the third and fourth weeks and to
delayed clearance of productive infection. In accordance with
previous findings (17), viral epitope-specific CD8 T cells per-
sisted in latently infected lungs, with a particularly high re-
sponse to the immediate-early epitope IE1, thought to be in-
volved in sensing of early stages in transcriptional reactivation
(43). Notably, although in the comparison between the two
viruses the replication of mCMV-WT.BAC was higher and
prolonged in the acute phase of infection, suggesting a lower
level of immune control, CD8 T-cell frequencies in latently
infected lungs were actually higher (Fig. 2B).
Previous work has revealed a chain of cause and effect,
relating virus titers during acute infection to the tissue load of
the latent viral genome, latency-associated viral transcription,
and the incidence of reactivation to productive infection (23,
34, 44). To verify this here for the role of vRAPs, we used the
established strategy of subdividing the lungs into 18 tissue
pieces (13, 22). Nine pieces of the three lobes of the right lung
were used for the analysis of IE1 transcription, two pieces of
the subcaval lobe were used for quantitating latent viral ge-
nome, and the seven pieces of the left lung were explanted to
determine the cumulative incidence of virus reactivation
Figure 3A (left panel) shows a much larger viral genome
load in lungs latently infected with WT virus, with only modest
interindividual and intratissue variance. This load difference
FIG. 3. Comparative molecular characteristics of viral latency established in the lungs. The analyses were performed at 6 months after BMT
and infection in the presence (gray-filled circles; WT.BAC) or absence (open circles; ?vRAP) of immunoevasins. (A) Comparison of latent viral
DNA loads determined by M55/gB gene-specific real-time quantitative PCR (qPCR) as described previously (43) and updated (42). Left panel:
data. Symbols show the viral DNA content of individually tested lung pieces no. 8 and 9 for five latently infected BMT recipients per group, 10
pieces per group altogether. Data were normalized to 106lung cells by pthrp gene-specific qPCR. The horizontal line indicates the mean value,
and the shaded area shows the 95% confidence interval for the mean. Right panel: statistical analysis. Shown is the “comparison cumulative fraction
plot” of the nonparametric and distribution-free Kolmogorov-Smirnov test for comparison of two data sets. In addition to providing a graphical
presentation of the data, this test has the advantage of making no assumption about the distribution of data. Instead, it compares the observed
empirical distributions, so that it is robust and applicable to all kinds of data sets. In addition, it also tests whether or not the data are normally
or log-normally distributed for applying parametric tests such as the Student t test. For a more detailed explanation, go to http://www.physics
.csbsju.edu/stats/KS-test.html. Indicated is the maximum difference (D value) between the cumulative fraction functions, also known as “empirical
distribution functions,” and the corresponding P value. A high D value and a low P value indicate a high difference between the data sets. The
difference is regarded as significant if the P value is ?0.05. Note that the cumulative fraction functions for the viral loads are completely separated
for the two viruses. Since the viral load data were found to follow a normal distribution, Student’s t test was also applied and showed a P value
of 0.001, with mean values of 4,091 and 501 for the latent genome loads of viruses WT.BAC and ?vRAP, respectively. (B) For lung pieces 10
through 18 of the same five mice, for 45 pieces per group altogether, the amounts of IE1 transcripts were determined by reverse transcriptase qPCR
as described previously (43), except that total lung tissue RNA was used as a template. Left panel: data, essentially as described above for panel
A. In addition, short horizontal bars indicate median values of data from individual mice. Negative data are indicated below the dotted line. Right
panel: statistical analysis. Unlike DNA load data, transcript data turned out not to be consistent with a normal or log-normal distribution. Shown
are the comparison cumulative fraction plot of the Kolmogorov-Smirnov test and the associated D and P values.
VOL. 83, 2009NOTES10295
FIG. 4. Virus reactivation in lung tissue explant cultures. (A) Characteristics of the assay. (A1) Functional stability of purified mCMV-WT.BAC
virions under standard cell culture conditions. Shown is the time course of activity loss. Symbols represent replicate cultures. The half-life (HL)
of infectivity is calculated from the negative slope (a) of the log-linear regression line according to the formula HL ? log102/a. Its 95% confidence
interval is indicated in parentheses. (A2) Time course of reactivated infection in four selected lung tissue explant cultures. Lung pieces derived
from BALB/c mice latently infected with mCMV-WT.BAC were plated directly into 24-well tissue culture plates with 1 ml of medium but with no
permissive feeder cells. At the indicated times, 0.1-ml aliquots of the culture supernatants were used for the PFU assay and replaced with fresh
medium. The first detection of infectivity is highlighted by gray shading. The dotted lines indicate the detection limit of the assay. (B) Comparative
reactivation distributions. Lung pieces no. 1 through no. 7 of the five mice per group, altogether 35 pieces per group, were plated at 6 months after
BMT and infection with the respective viruses. (B1) Illustration of the time course of reactivations observed in the explant cultures. Results are
shown as binary data. Open circles, no reactivation; black dots, reactivation. (B2) Cumulative reactivation incidence plot. It is important to
understand that according to the Poisson distribution of reactivations in each explant, a positive explant culture may reflect more than one
reactivation, so that the actual number of reactivations exceeds the observed number of positive cultures. For each time point, the total number
of reactivations was calculated from the observed fraction of negative cultures [f(0)], which equals e??, according to the formula: ? F(n) ? ?/n ?
F(n ? 1) for all n values of ?0, where ? is the Poisson distribution parameter lambda and [F(n)] is the fraction of cultures comprising a number
(n) of reactivations. (B3) Statistical analysis. Shown is the comparison cumulative fraction plot of the Kolmogorov-Smirnov test. The maximum
difference, D, between the cumulative fraction functions and the corresponding P value is indicated.
turned out to be highly significant, as revealed by Kolmogorov-
Smirnov statistics (Fig. 3A, right panel) and by Student’s t test.
Likewise, IE1 transcription was also elevated in lungs latently
infected with WT virus (Fig. 3B, left panel). In agreement with
previous data on a Poisson distribution of transcripts (13), the
data here were neither normally nor log-normally distributed.
Again, the distribution-free Kolmogorov-Smirnov statistics re-
vealed a highly significant difference between the two groups
(Fig. 3B, right panel). Thus, apparently, immunoevasins have
an impact on latent viral DNA load and on viral transcriptional
activity during latency.
The definition of herpesvirus latency by Roizmann and Sears
(41) demands that the viral genomes not only are physically
maintained but are reactivatable to productive infection. Since
reactivation is a stochastic process, approaches of in vivo re-
activation can give only the “point prevalence” of reactivation
for the time of analysis (23). We have here therefore chosen
the long-established method of tissue explantation (21) to
measure the cumulative reactivation incidence over time,
which gives a better estimate for the number of reactivatable
latent genomes (Fig. 4). Figure 4A introduces the experimental
system by showing exemplarily the half-life of infectious
mCMV-WT.BAC virions in cell culture medium (Fig. 4A1), as
well as differences in the onset of reactivated infection in lung
explants (Fig. 4A2). Importantly, the presence of reactivated
virus in explant culture supernatants by far exceeded the half-
life of virions. Specifically, explanted tissue supported infection
for an extended period of ?2 months until it gradually got
exhausted. Accordingly, an explant culture, once positive, re-
mained positive throughout the reactivation assay (Fig. 4B).
Figure 4B1 sketches reactivation events as virus-positive cul-
tures observed over time until no further reactivation was
observed. At a glance, reactivation occurred frequently from
lung pieces carrying latent WT virus but rarely from pieces
carrying the latent mutant virus. As shown in the cumulative
plot of reactivation events over time, most reactivations oc-
curred between 2 and 4 weeks in culture, which proves that
lung pieces were not productively infected at the time of ex-
plantation (Fig. 4B2). The difference between the two groups
was highly significant as revealed by Kolmogorov-Smirnov sta-
tistics (Fig. 4B3). In total, for WT virus, we determined 31 virus
reactivations from 35 explants. Since an explant contains ?3
million cells with a load of ?4,000 genomes per million cells
(recall Fig. 3A), the cumulative reactivation incidence was in
the range of 10?4.
The difference in the reactivation incidences most likely
reflects the difference in the latent viral DNA load (34). As
shown recently, although deletion of the regulatory protein
IE1 attenuates mCMV (10), an IE1 deletion mutant was still
able to reactivate provided that genome loads of WT and
mutant virus were adjusted by using higher doses of mutant
virus for acute infection (5).
Notably, the latency parameters load, transcription, and re-
activation were here found to be positively correlated with the
magnitude of the immune response. This paradox may be ex-
plained by the previously described negative feedback regula-
tion of CD8 T-cell stimulation (4). According to this model,
the expression of immunoevasins inhibits the recognition of
infected cells and thus the control of infection, thereby pro-
moting a sustained antigen supply for cross-priming of the
CD8 T-cell response by uninfected antigen-presenting cells.
Although such a mechanism was originally proposed for CD8
T-cell priming in the regional lymph node, it may also apply to
effector memory cells in latently infected lungs.
This work was supported by the Deutsche Forschungsgemeinschaft,
SFB 490, individual projects E2 (C.K.S. and C.O.S.), E3 (R.H.), and
E4 (V.B. and M.J.R.), and Clinical Research Group KFO 183, indi-
vidual project TP8 (M.J.R.).
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