Vaccination against a hit-and-run viral cancer
Philip G. Stevenson, Janet S. May, Viv Connor and Stacey Efstathiou
Philip G. Stevenson
Received 11 May 2010
Accepted 21 June 2010
Division of Virology, Department of Pathology, University of Cambridge, UK
Cancers with viral aetiologies can potentially be prevented by antiviral vaccines. Therefore, it is
important to understand how viral infections and cancers might be linked. Some cancers
frequently carry gammaherpesvirus genomes. However, they generally express the same viral
genes as non-transformed cells, and differ mainly in also carrying oncogenic host mutations.
Infection, therefore, seems to play a triggering or accessory role in disease. The hit-and-run
hypothesis proposes that cumulative host mutations can allow viral genomes to be lost entirely,
such that cancers remaining virus-positive represent only a fraction of those to which infection
contributes. This would have considerable implications for disease control. However, the hit-and-
run hypothesis has so far lacked experimental support. Here, we tested it by using Cre–lox
recombination to trigger transforming mutations in virus-infected cells. Thus, ‘floxed’ oncogene
mice were infected with Cre recombinase-positive murid herpesvirus-4 (MuHV-4). The emerging
cancers showed the expected genetic changes but, by the time of presentation, almost all lacked
viral genomes. Vaccination with a non-persistent MuHV-4 mutant nonetheless conferred
complete protection. Equivalent human gammaherpesvirus vaccines could therefore potentially
prevent not only viral genome-positive cancers, but possibly also some cancers less suspected of
a viral origin because of viral genome loss.
(Blumberg, 1997) and cervical (Frazer, 2004) cancers has
made antiviral vaccination a relatively simple and effective
means of disease prevention. Human gammaherpesviruses –
Epstein–Barr virus (EBV) and Kaposi’s sarcoma-asso-
ciated herpesvirus (KSHV) – are also oncogenic, but
the lack of single, unifying features of the associated
cancers has made it unclear how directly infection and
disease are linked and so what vaccination might achieve.
The robust persistence of herpesviruses in immunocom-
petent hosts also makes vaccination a considerable
EBV transforms B cells in vitro and, in immunocomprom-
ised patients, the viral genes responsible for transformation
can cause disease (Carbone et al., 2009). However, EBV-
infected cancers in immunocompetent hosts tend to
express the same viral genes as non-transformed cells.
They differ in also carrying oncogenic host mutations;
indeed, Burkitt’s lymphoma is associated more strongly
with c-myc translocation than with EBV infection
(Thorley-Lawson & Allday, 2008). Thus, viral genes seem
mostly to have triggering or accessory roles in disease, with
host oncogenes being the main drivers. The hit-and-run
hypothesis proposes that viral genomes initiating disease
can be lost entirely to obscure a cancer’s viral origin
(Ambinder, 2000). Early on, viral genes are likely to be
essential for cancer-cell survival (Hammerschmidt &
Sugden, 2004). However, cancers accumulate vast numbers
of host mutations (Pleasance et al., 2010), some of which
will inevitably promote more autonomous growth. Thus, it
seems inevitable that a cancer will, with time, evolve
increasing independence from viral gene functions that
could allow viral genome loss.
The main problem with the hit-and-run hypothesis has been
a lack of experimental support. Analyses of gammaherpes-
virus-induced cancers have focused on African Burkitt’s
plausible a causal link between infection and disease.
However, focusing on virus-positive cancers tells us little
about genome loss, as here most presenting cancers would be
virus-negative. Instead, it is necessary to track prospectively
the fate of viral genomes in transformed cells. In vitro, B-cell
cancers tend to maintain gammaherpesvirus genomes,
whereas Kaposi’s sarcoma and nasopharyngeal carcinoma
murid herpesvirus-4 (MuHV-4) infection increases the
incidence of virus-negative cancers (Sunil-Chandra et al.,
1994; Tarakanova et al., 2005). However, the difficulty of
analysing spontaneous cancers, where the molecular changes
driving transformation are almost always unknown, makes
firm functional conclusions hard to draw. To ensure that the
host factors contributing to cancer remained known, we used
Cre–lox recombination in a well-established conditional
mouse cancer model (reviewed by DuPage et al., 2009) to
cancers for viral genome retention.
Journal of General Virology (2010), 91, 2176–2185
2176023507G2010 SGM Printed in Great Britain
Generation of Cre+MuHV-4
We inserted a human cytomegalovirus (HCMV) IE1
promoter-driven Cre expression cassette between the 39
ends of MuHV-4 ORFs 57 and 58 (Fig. 1a, b). We used an
HCMV IE1 promoter because this can be active in latently
infected cells (Rosa et al., 2007; Smith et al., 2007). Thus,
Cre could be expressed without MuHV-4 lytic genes kill-
ing the infected cells. Two functionally indistiguishable
mutants were obtained. Both showed Cre expression by
excising spontaneously their loxP-flanked bacterial artificial
chromosome (BAC) cassettes, and immunofluorescence
showed Cre expression in infected-cell nuclei (Fig. 1c).
(The Cre coding sequence used incorporates an N-terminal
In vivo loxP recombination by Cre+MuHV-4
We tested whether viral Cre expression could recombine
loxP sites in the host genome by infecting mouse
embryonic fibroblasts derived from ROSA26-lacZflox/flox
reporter mice (Fig. 2a). b-Galactosidase assays were
strongly positive, indicating loxP recombination. Such
recombination was also achieved by infecting ROSA26-
lacZflox/floxmice intraperitoneally (i.p.) with Cre+MuHV-
4 (Fig. 2b): widespread b-galactosidase expression was
evident on the diaphragm, a site commonly infected by i.p.
MuHV-4 (Milho et al., 2009).
We then infected p53flox/floxK-rasLSL-G12D/+mice i.p. with
Cre+MuHV-4 (Fig. 2c, d). More than 90% of infected
mice developed cancers within 3 months, compared with
0% of uninfected or wild-type MuHV-infected controls.
Cancers occurred most frequently on the diaphragm.
Disease was rare within 30 days, and most cancers were
single lesions. In contrast, virus replication was widespread:
3 days after inoculation, spleens yielded (2.1±1.2)6104
and peritoneal washes (1.7±1.2)6103infectious centres
per mouse (mean±SD titres, n56, with lytic titres ,1% of
infectious centre titres); even 2 months later, spleens
yielded (2.2±1.5)6102infectious centres per mouse
(n56). Therefore, cancer growth was much more restricted
than viral latency and functional Cre expression.
Analysis of virus-triggered cancers
All of the cancers analysed (n.12) were histological
sarcomas (Fig. 3a). In situ hybridization (Fig. 3b) showed
surprisingly littleexpression oftheMuHV-4tRNAsnormally
abundant in lytic and latent infections (Bowden et al., 1997).
At most, a few positive cells were scattered around the main
cancer mass. Real-time PCR (Fig. 3c) established that
sarcomas contained lower copy numbers of viral genomes
than latently infected spleens of the same mice.
Fig. 1. Characterization of Cre+MuHV-4. (a) An HCMV IE1 promoter-driven Cre expression cassette was inserted between
MuHV-4 ORFs 57 and 58. Relevant restriction sites are shown. (b) Viral DNA was digested with HindIII or BglII and probed
with either a genomic BglII clone or the HCMV IE1–Cre construct, as shown in (a). WT, Wild-type; Cre+, recombinant;
Cre+ind, independently derived recombinant. (c) BHK-21 cells were infected with wild-type or Cre+MuHV-4 (1 p.f.u. per cell,
16 h), then fixed, permeabilized and stained for Cre recombinase or for MuHV-4 antigens using polyclonal rabbit sera. Nuclei
were counterstained with DAPI.
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Viral genome loss