JOURNAL OF VIROLOGY, June 2006, p. 5383–5387
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 11
Effect of Apolipoprotein E on the Cerebral Load of Latent
Herpes Simplex Virus Type 1 DNA
Javier S. Burgos,†* Carlos Ramirez,† Isabel Sastre, and Fernando Valdivieso*
Departamento de Biologı ´a Molecular and Centro de Biologı ´a Molecular Severo Ochoa (CSIC-UAM),
Universidad Auto ´noma de Madrid, Madrid, Spain
Received 2 January 2006/Accepted 10 March 2006
Herpes simplex virus type 1 (HSV-1) is neurotropic and enters a latent state lasting the lifetime of the host.
This pathogen has recently been proposed as a risk factor for Alzheimer’s disease (AD) in conjunction with
apolipoprotein E4 (ApoE4). In a murine acute infection model, we showed that viral neuroinvasiveness depends
directly on the overall ApoE dosage and especially on the presence of isoform ApoE4. If an interaction between
ApoE and HSV-1 is involved in AD, it may occur during latency rather than during acute infection. Certainly,
ApoE plays an important role in late-onset AD, i.e., at a time in life when the majority of people harbor HSV-1
in their nervous system. In the present work, wild-type, APOE knockout, APOE3, and APOE4 transgenic mice
were used to analyze the influence of the ApoE profile on the levels of latent virus DNA. The knockout mice
had significantly lower concentrations of the virus in the nervous system than the wild-type mice, while
the APOE4 mice had very high levels in the brain compared to the APOE3 animals. ApoE4 seems to
facilitate HSV-1 latency in the brain much more so than ApoE3. The APOE dosage correlated directly with
the HSV-1 DNA concentration in the brain, strengthening the hypothesis that HSV-1, together with ApoE,
might be involved in AD.
Most cases of Alzheimer’s disease (AD) (?95%) are late
onset, are of the sporadic type, and appear to be associated
with no known genetic markers (22). A variety of potential risk
factors, both genetic and environmental, have been examined,
but the etiology and pathogenesis of sporadic AD are still
poorly understood. The idea that both kinds of factors interact
and contribute to the development of AD is gaining wide
acceptance. Although many proposed causes have been stud-
ied, only two factors are widely accepted as being associated
with nonfamilial disease: age (16, 19, 35, 38) and the possession
of the apolipoprotein E ε4 allele (APOE4) (4, 41, 50).
Apolipoprotein E (ApoE), a major component of very-low-
density lipoproteins, may play an important modulatory func-
tion in the central nervous system (CNS): in the peripheral
nervous system, several apolipoproteins are involved in lipid
transport, but in the CNS, there is less redundancy (32, 40).
ApoE is involved in the mobilization and redistribution of
lipids and cholesterol during neuronal growth and repair (12,
42), the long-distance systemic and cerebrospinal transport of
lipids (17), and the promotion of synaptic plasticity following
neuronal injury (12). The human APOE gene has three com-
mon alleles, ε2, ε3, and ε4, which encode the ApoE2, ApoE3,
and ApoE4 protein isoforms, respectively (43). Sequencing
studies have shown ApoE3 and ApoE4 to differ by just a single
amino acid (Cys or Arg at residue 112, respectively) (48, 52).
APOE3 is the most common allele. APOE4 has been associ-
ated with a high risk for AD in many studies, irrespective of
race or geographical location (10, 17, 28, 31, 37). Nevertheless,
the APOE4 allele alone is neither necessary nor sufficient for
the development of AD (13, 44).
Although the involvement of an infectious agent in the eti-
ology of AD is far from fully demonstrated, herpes simplex
virus type 1 (HSV-1) has recently been implicated in the patho-
genesis of this disease (22, 25, 34). Several coincidences rein-
force this association: HSV-1 is neurotropic (in line with the
neurological nature of AD), is ubiquitous in the human pop-
ulation (36), and infects and causes pathological changes in the
brain regions mainly affected by AD (1, 2). One of the impor-
tant features of this virus is its ability to establish a latent
infection and to reside in the nervous system over the lifetime
of the host. A prominent characteristic of the virus is its ability
to reactivate from latency and cause viral shedding and/or
recurrent disease. HSV-1 has been found in the brains of many
patients with AD but has also been found in normal brains of
elderly people (25, 26), suggesting that HSV-1 infection is not
independently associated with AD (22). However, while the
incidence of AD is not increased in those with HSV-1 DNA or
the APOE4 allele alone (25), it is highest in the carriers of this
allele who also harbor HSV-1 DNA in the CNS.
Using a murine model of acute hematogenous HSV-1 infec-
tion, we previously reported that viral neuroinvasion was re-
duced in mice lacking ApoE compared to wild-type mice and
that the ApoE dose was directly linked to the HSV-1 DNA
concentration detected in the nervous system (8). In a further
study, we showed that during acute infection, ApoE4 was more
efficient than ApoE3 in promoting colonization of the brain by
HSV-1 (comparisons were made at the time when this organ
presented the highest viral loads) (7). If an interaction between
ApoE and HSV-1 has anything to do with AD, this may occur
HSV-1 DNA may be present in the majority of elderly hu-
man brains (26). Almost all adults show evidence of having
* Corresponding author. Mailing address: Lab CX340, Centro de
Biologı ´a Molecular, Universidad Auto ´noma de Madrid, Cantoblanco,
28049 Madrid, Spain. Phone: 34 91 4978471. Fax: 34 91 4974870.
E-mail for Javier S. Burgos: firstname.lastname@example.org. E-mail for Fernando
† J.S.B. and C.R. contributed equally to this work.
been infected by HSV-1, its genome residing latently in the
trigeminal ganglia and in the CNS of both healthy individuals
and those suffering from neurological disease (14). The trigem-
inal ganglion is the primary site of HSV-1 latency, although
other sites including the sensory neurons (33) and other sen-
sory ganglia such as the nodose ganglion of the vagus nerve
(18), the dorsal root ganglia (20), the sympathetic ganglia (51),
and the brain may be involved (3, 47). The persistence of
HSV-1 in nonneuronal tissue has also been suggested but re-
mains controversial (33).
Latency is a poorly understood phenomenon, and the host
factors that contribute to its establishment and maintenance
are far from fully known. The present work describes how the
ApoE profile affects the levels of latent HSV-1 and shows that
ApoE4 in particular is associated with increased amounts of
latent virus in the brain. The relationship between these two
risk factors for AD could be of great importance in the patho-
genesis of this disease.
MATERIALS AND METHODS
Inoculation and dissection. All experiments were performed in accordance
with the guidelines of the 1986 European Community Animals Act (Scientific
Procedures). All animals underwent a period of quarantine. Strict precautions
were taken to prevent contamination during inoculation and dissection.
The animals used were 37 14-week-old C57BL/6 female mice: 19 wild-type mice
(Harlan, Barcelona, Spain), 6 APOE knockout mice, 6 APOE4 human transgenic
mice, and 6 APOE3 human transgenic mice. All mice were marked, examined, and
using a protocol described previously by Piedrahita et al. (39), were obtained
from Taconic M&B (Bomholtrej, Denmark). To compare the isoform-specific
effects of ApoE3 and ApoE4 on HSV-1 latent infection, mice transgenic for
human APOE3 (B6.129P2-ApoeTm1(APOE3)MaeN8) and APOE4 (B6.129P2-
ApoeTm1(APOE4)MaeN8) were purchased from the same supplier. These mice
were generated by replacing their APOE gene with the corresponding human
homolog as described previously by Knouff et al. and Sullivan et al. (30, 49). Such
animals are useful for in vivo studies of the human ApoE isoforms since the
replacement of the mouse APOE does not alter the murine regulatory sequence.
Consequently, the mice express the human ApoE isoforms with tissue distribu-
tions and levels very close to those of endogenous murine ApoE.
HSV-1 was propagated and titrated by plaque assay in confluent monolayers
of Vero cells (8). HSV-1 strain KOS (kindly supplied by L. Carrasco) was used
in all experiments. Female mice were intraperitoneally inoculated with doses of
105, 106, or 107PFU of virus suspension as previously described (6). The mouse
strain and the female gender were selected in order to compare the results with
previously published data (6–8). The female gender was also used because this
sex shows greater viral infectivity than males (6). An extra set of mock-infected
animals was used as controls. Since overt infection subsides 2 weeks after inoc-
ulation and all HSV-1 present is in a latent state by 4 weeks after inoculation
(33), the mice were sacrificed at 37 days postinfection (when latent infection was
assured), and their organs were carefully removed and frozen at ?70°C. The
HSV-1 DNA concentrations in whole blood, the adrenal glands, the gonads, the
spinal cord, the trigeminal ganglia, and the brain were then determined. For
more precise analyses, the brain samples were subdivided into four regions: the
midbrain (including the midbrain and nearby structures such as the pons, the me-
dulla, and the superior and inferior colliculi), the ventricles (including the third and
lateral ventricles, the thalamus, the hypothalamus, the preoptic area, and the
striatum), the cerebral cortex (including the cortex; the temporal, frontal, pari-
etal, and occipital lobes; the hippocampus; the corpus callosum; and the olfactory
bulbs), and the cerebellum.
HSV-1 DNA quantification in tissue homogenates. DNA from homogenized
samples was extracted by conventional methods (DNeasy 96 tissue kit [QIAGEN
Sciences, MD] or QIAamp 96 DNA blood kit [QIAGEN GmbH, Hilden, Ger-
many]). The concentration of HSV-1 DNA in different organs was then quanti-
fied by real-time quantitative PCR with an ABI Prism 7900HT SD system
(Applied Biosystems) using a Custom TaqMan assay (a specific assay for a
sequence belonging to the US12 viral gene). Reactions were performed under
universal conditions using TaqMan Universal PCR master mix (Applied Biosys-
tems). The exact same PCR protocol was used to quantify the mouse genomic
DNA using an Assay-On-Demand probe specific for the GAPDH (glyc-
Mm99999915_g1; Applied Biosystems). This generated an amplicon of 107 bp
from the GenBank accession number NM_001001303.1 transcript. An appropri-
ate concentration range of virus was used for the optimization of the standard
curve; the viral DNA concentration was expressed in terms of viral copy num-
bers. PCR calibration was performed by amplification of the GAPDH house-
keeping gene from a concentration range of mouse genomic DNA (results are
expressed as nanograms of host DNA). Viral DNA loads were also corroborated
by amplification of the thymidine kinase viral gene and the ?-actin housekeeping
gene by real-time PCR using a LightCycler rapid thermal cycler (Roche Diag-
nostics Ltd., Lewes, United Kingdom) and a LightCycler FastStart DNA Master
SYBR Green I kit (catalog no. 3 003 230; Roche, Germany) as previously
described (6–9). Each experiment was performed in triplicate. Melting curve
analyses, agarose and acrylamide gel electrophoresis, restriction analysis, and
nested PCR confirmed the specificity of the products (5, 9).
Statistical analysis. Fisher’s exact test was used to compare viral DNA con-
centrations. Significance was set at a P value of ?0.05.
Effect of viral inoculation dose on latent HSV-1 DNA levels.
To evaluate the influence of the injected viral dose on the final
concentrations of latent viral DNA, wild-type female mice
were inoculated with between 105and 107PFU. Figure 1 shows
the viral DNA load of several organs in relation to the admin-
istered dose. The HSV-1 DNA concentration was always di-
rectly dependent on the inoculation dose, with a similar pat-
tern of infection seen for all doses analyzed. Independent of
the dose, the organs with the highest HSV-1 DNA levels were
the nervous system (spinal cord, brain, and trigeminal ganglia,
where the virus establishes latency) and the blood (where
amounts of virus produced during latency eventually arrive
from the trigeminal ganglia) (6). The viral DNA levels of the
four brain regions analyzed (Fig. 1, right inset) were also in-
oculation dose dependent. The midbrain had the highest viral
DNA load for every dose tested, followed by the ventricles,
FIG. 1. Quantification of viral DNA loads during latency with re-
spect to the viral dose injected into wild-type mice. The bar graph
represents the viral copy number detected in each organ expressed on
a logarithmic scale. The inset shows the areas of the brain analyzed.
Values are the means ? standard errors of the means (SEM) of the
quantity of viral DNA (expressed as viral genomes and normalized
with respect to the quantity of mouse genomes in 100 ng of host DNA).
Fisher’s exact test was used to compare the HSV-1 DNA concentra-
tions achieved with the different doses (always compared to a dose of
106PFU) (*P ? 0.05).
5384 BURGOS ET AL.J. VIROL.
cortex, and cerebellum. This order was consistent in all exper-
iments. For further experiments, an inoculation dose of 106
PFU per animal was selected.
Effect of ApoE on HSV-1 latent infection. At 37 days postin-
fection, HSV-1 was preferentially detected in the nervous sys-
tem and in the whole blood of the wild-type (or APOE?/?)
mice; the ovaries and adrenal glands showed lower levels of
viral DNA. When DNA loads in the different organs of the
APOE?/?and APOE knockout (or APOE?/?) mice were
compared, those of the wild-type mice showed significantly
higher concentrations, except for whole blood (Fig. 2). In both
backgrounds, the organ with the highest viral DNA load after
the blood was the brain, indicating preferential viral tropism
towards this organ and the establishment of latency in the
CNS. The differences between HSV-1 DNA concentrations in
APOE?/?and APOE?/?mice were dramatic in the brain
(wild-type-to-knockout ratio, 13.7; P ? 0.001), spinal cord (ra-
tio, 52.1; P ? 0.001), and trigeminal ganglia (ratio, 23.7; P ?
0.001). In the dissected brains of APOE?/?mice, the order of
viral DNA concentrations was midbrain, ventricles, cortex, and
cerebellum. APOE?/?mice had detectable levels of viral DNA
only in the midbrain but at considerably lower concentrations
(ratio, 11.7; P ? 0.001), and no viral DNA was detected in the
distal regions of their brains (Fig. 2, inset). The concentra-
tions in the ovaries and adrenal glands, although very low,
were significantly different (P ? 0.05) in the wild-type and
Effect of ApoE isoform on HSV-1 latent infection. Striking
isoform-dependent differences were seen in the viral DNA
concentrations of the infected organs (Fig. 3). In general,
higher viral DNA loads were detected in the organs of APOE4
mice than in the organs of APOE3 mice. The blood and the
nervous system were the sites where the virus was preferen-
tially detected. The nervous system viral DNA concentrations
showed significant genotype-dependent differences; the brain
APOE4-to-APOE3 viral DNA ratio was 13.6 (P ? 0.001), that
of the spinal cord was 180.7 (P ? 0.001), and that of the
trigeminal ganglion was over 200 (P ? 0.001). Differences in
brain region viral DNA loads were found between APOE4 and
APOE3 mice (Fig. 3, inset). HSV-1 DNA levels in the ventri-
cles, cortex, and cerebellum of APOE3 mice were undetect-
able, and only low levels were found in the midbrain of APOE3
mice compared to those of APOE4 mice (ratio, 12.7; P ?
0.001). However, no significant differences were seen between
these groups with respect to blood and adrenal gland viral
DNA loads. In conclusion, the present data show that HSV-1
DNA levels during viral latency in the brain are more than 10
times higher in the APOE4 background than in the APOE3
background. Interestingly, the differences seen between the
APOE4 and APOE3 mice were comparable to those observed
between APOE?/?and APOE?/?animals (compare Fig. 2
and 3). The human APOE3 genetic background seemed to
reduce the establishment of latency in a manner similar to the
lack of APOE. Finally, in the majority of organs, the APOE4
and wild-type mice differed very little with respect to HSV-1
DNA concentrations. Moreover, the order of viral DNA levels
in the different brain regions of these mice was identical.
HSV-1, one of the most commonly encountered pathogens,
has been related to several neurological diseases, especially
AD (23, 25, 34). Infection with this virus, which can on occa-
sion cause long-term neurodevelopmental damage in neo-
nates, is characterized by the pathogen entering a latent state
in the nervous system that lasts the lifetime of the host. The
foremost site for this latent infection is the trigeminal ganglia,
a location very close to the brain region where the protein
tangles associated with AD first appear. Interestingly, HSV-1
FIG. 2. Quantification of viral DNA levels in relation to APOE
dosage. Black bars represent the wild-type mice, and white bars rep-
resent the APOE knockout group (all mice were inoculated with 106
PFU of HSV-1). The inset shows the areas of the brain analyzed.
Values are the means ? SEM of the quantity of viral DNA (expressed
as viral genomes and normalized with respect to the quantity of mouse
genomes in 100 ng of host DNA). Fisher’s exact test was used to
compare the values for the different APOE dosage groups (*P ? 0.05).
FIG. 3. Quantification of HSV-1 DNA concentrations related to
the ApoE isoform. Black bars represent the APOE4 group, and white
bars represent the APOE3 group (all animals were injected with 106
PFU of HSV-1). The inset shows the areas of the brain analyzed.
Values are the means ? SEM of the quantity of viral DNA (expressed
as viral genomes and normalized with respect to the quantity of mouse
genomes in 100 ng of host DNA). Fisher’s exact test was used to
compare the values for the different ApoE isoform groups (*P ? 0.05).
VOL. 80, 2006ApoE AND HSV-1 LATENCY5385
DNA is detectable in regions of the brain that are affected by
this disease (2).
We previously reported that acute infection of the brain by
HSV-1 is influenced by the ApoE dosage and isoform (6–8).
However, if HSV-1 is truly involved in AD, it may be associ-
ated with latency rather than acute infection. The establish-
ment of the latent state is regulated by viral and host factors
(both cellular and immune), not all of which are known or
understood (15); the present results suggest, however, that the
ApoE dosage and isoform play a critical role.
HSV-1 becomes latent in the neurons of both the peripheral
nervous system and the CNS (11). Although the trigeminal
ganglia represent the primary site where HSV-1 resides in the
latent form (33), other locations have been documented, in-
cluding the nodose ganglion of the vagus nerve (18), the dorsal
root ganglia (20), the sympathetic ganglia (51), and the brain
(3, 47). Thus, the spinal cord and brain could act as reservoirs
of latent virus. Most, if not all, human brains harbor latent viral
genomes (14, 26, 27), but their quiescence or, conversely, their
ability to cause harm may depend on their exact location, the
immune status of the host, and their copy number (in which
ApoE could play an important role). It has been shown that the
HSV-1 genome copy number is positively correlated with the
ability to reactivate in vivo in neurons of the trigeminal ganglia
(46). After reactivation, recurrent disease may develop in neu-
rons harboring the latent virus; in fact, HSV-1 has been impli-
cated in necrotic and apoptotic neural cell death in the trigem-
inal ganglia of acutely infected experimental animals (29, 53).
Recently, we provided direct evidence that HSV-1 reactivates
molecularly in the hippocampal neurons (5); this could be the
cause of the local degenerative neuropathological complex that
Possession of the APOE4 allele has been associated with the
development of AD (10, 45). However, this alone is neither
essential nor sufficient; interaction with another factor appears
to be necessary. Itzhaki et al. previously showed that the rela-
tive risk of developing AD for those positive for HSV-1 DNA
in the brain and who carried an APOE4 allele was considerably
higher than that for individuals with only one (or none) of
these factors (22, 24, 25). In addition, Itabashi et al. previously
showed that patients with AD were more likely to be positive
for both HSV and the APOE4 allele than for one factor alone
(21). These results suggest that the combination of these two
factors is strongly associated with the development of AD.
However, the latter studies do not show how this ApoE iso-
form influences infection or predisposes those infected to AD,
and until now, it was unclear whether a direct correlation
between viral DNA levels in the brain and the possession of the
APOE4 allele existed. ApoE4, perhaps unlike other ApoE
isoforms, may increase the risk of AD by increasing HSV-1
viral loads in the nervous system.
The present results show that ApoE plays an important role
in latent virus DNA concentrations. In APOE-deficient mice,
HSV-1 DNA levels were significantly lower than those in wild-
type mice not only in the brain (13-fold) but in every organ
analyzed, except for whole blood. Furthermore, the APOE
allele possessed by infected mice influenced the efficiency of
the establishment (or maintenance) of latent infection: the
encephalons of APOE4 animals contained significantly more
viral DNA (13-fold) than those of APOE3 mice. Interestingly,
the viral DNA ratios seen for APOE?/?:APOE?/?and
APOE4:APOE3 mice were identical and comparable to the
ratios observed in acute infection (7, 8). This agrees with the
finding that individuals positive for HSV-1 DNA in the brain
and who carry the APOE4 allele are 12 times more likely to
develop AD than those possessing only one (or neither) factor
(24). The present results regarding the effect of ApoE dosage
(presence compared to absence) on latent HSV-1 infection of
the brain also agree with the generally accepted dose effect
reported for APOE4 in AD (10). In the establishment of latent
infection, the APOE?/?and APOE4 genotypes were associ-
ated with high HSV-1 DNA concentrations in the nervous
system, while APOE?/?and APOE3 were associated with low
This work is the first to show that latent cerebral HSV-1
infection depends on the APOE profile. This may be important
in the onset of AD since the incidence of this disease has been
related to the possession of the APOE4 allele and the presence
of HSV-1 DNA in the CNS; it is possible, therefore, that these
agents are cofactors that promote the disease. This new evi-
dence strongly reinforces the idea that HSV-1 is an etiological
factor in AD.
This work was supported by a grant from the Obra Social Caja
Madrid to the Asociacio ´n de Familiares de Enfermos de Alzheimer
and by an institutional grant from the Fundacio ´n Areces to the Centro
de Biologı ´a Molecular Severo Ochoa.
We thank F. Mayor for his continuous encouragement and help and
L. Carrasco for providing the HSV-1 KOS strain.
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