442

Cosmid-based mutagenesis and methods to examine

varicella-zoster virus (VZV) tropism for differentiated human
cells in vivo provide new information about molecular
mechanisms of VZV infection. How specific VZV gene
products contribute to viral replication has been further
defined, and effects of VZV on expression of cellular genes
have been demonstrated.
Addresses
G-312, Stanford University School of Medicine, 300 Pasteur Drive,
Stanford, California 94305, USA; e-mail: aarvin@stanford.edu
Current Opinion in Microbiology 2001, 4:442449
1369-5274/01/$ see front matter
2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
DC dendritic cells
ER endoplasmic reticulum
g glycoprotein
GFP green fluorescent protein
HCMV human cytomegalovirus
HIV human immunodeficiency virus
HSV-1 herpes simplex virus 1
I FN- interferon-
NK natural killer
ORF open reading frame
PBMC peripheral blood mononuclear cell
PHN post-herpetic neuralgia
P- Oka parent Oka
TBP TATA-binding protein
TG trigeminal ganglia
TGN trans-Golgi network
TNF- tumor necrosis factor-
V- Oka vaccine Oka
VZV varicella-zoster virus
Introduction
Varicella-zoster virus (VZV) is one of eight human her-
pesviruses; by new nomenclature, it is designated HHV-3
and is classified in the alpha-herpesvirus subfamily. On the
basis of the linear arrangement of its 69 open reading
frames (ORFs), along the double-stranded DNA genome
and sequence similarities within the homologous ORFs,
VZV is related most closely to herpes simplex virus 1
(HSV-1). VZV causes varicella (chicken pox) during primary
infection and establishes latency in dorsal root ganglia;
reactivation from latency results in herpes zoster (shingles).
In the course of infection, VZV exhibits tropism for T
lymphocytes, causing viremia, as well as for skin and neuronal
and non-neuronal cells of the sensory ganglia. VZV is the
first human herpesvirus for which live attenuated vaccines,
derived from the Oka strain (vaccine Oka; V-Oka), have
been licensed. Because VZV is a significant human
pathogen, new insights about virushost interactions have
emerged from clinical as well as basic investigations. This
review highlights new information about basic virology,
pathogenesis and host response to VZV.
Genetic analysis
Although geographic differences are evident by restriction
enzyme analysis, the VZV genome has been considered
to be highly stable, with little antigenic variability or
virulence differences expected among wild-type isolates.
For example, the epitope stability of the predominant
glycoprotein E (gE) was demonstrated using a panel of
monoclonal antibodies to test unrelated VZV isolates at
low and high passages (i.e., the virus recovered from the
patient was first cultured in tissue culture cells, then trans-
ferred to uninfected cells a few times [low passage] or
multiple times [high passage]) [1]. However, the analysis
of the VZV-MSP strain demonstrates the exception, as this
clinical isolate has an accelerated replication phenotype
that is attributable to one amino acid substitution in gE,
encoded by ORF68 [2

].
Sequence changes in V-Oka are relevant for understanding its
attenuation in the human host, which has been established
through extensive clinical experience [3,4]. Whereas the
genetic basis for the reduced virulence phenotype of V-Oka
is not known, sequence comparisons with its parent strain
(P-Oka) and other VZV isolates have been reported
[3,5,6

]. These analyses reveal nucleotide differences

between V-Oka and other VZV isolates that alter amino
acid residues in all classes of viral proteins. For example,
ten single-nucleotide polymorphisms (SNPs) are present
in ORF62 and several are detected in the viral glycoprotein
genes, including gE [5]. Whereas subclones of P-Oka are
shown to be consistent by sequencing, subpopulations are
identified in varicella vaccine that have variations in the
ORF62 gene, which encodes the IE62 protein, the major
immediate early transactivator [6

]. Our comparative
analysis of the V-Oka and P-Oka consensus sequence data
is accessible on the Internet [7]. How these V-Oka sequence
differences modulate virushost interactions will require
careful investigation because the analysis of VZV-MSP
shows that even single base-pair changes have the potential
to alter VZV infectivity in cell culture and in vivo, in the
SCIDhu-mouse model of VZV pathogenesis [2

].
Identifying stable sequence changes is important to allow
differentiation of V-Oka from circulating wild-type VZV
strains. V-Oka has differences in the ORF62 gene and in
the ORF64 poly-A region that can be used for this purpose;
these changes were stable even when V-Oka was transmitted
from a vaccine recipient and replicated in a susceptible
contact [3]. PCR amplification and restriction enzyme
analysis of a region of ORF62 distinguished V-Oka from
Varicella-zoster virus: molecular virology and virushost
interactions
Ann M Arvin
wild-type VZV strains recovered from all geographic areas,
including Japan, where V-Oka was first derived [4].
A direct effect of genetic mutations on VZV infectivity was
demonstrated by deriving isolates resistant to the antiviral
drug foscarnet [8]. Drug exposure selected for mutants
that had single base-pair changes in the DNA polymerase
gene that resulted in amino acid substitutions. Variable
mutations occurred, altering domains II or III of the DNA
polymerase, and were associated with a slow growth phe-
notype and delayed expression of immediate early proteins.
That foscarnet-resistant subpopulations were recovered
from seven wild-type isolates means that drug-resistant
VZV may emerge during antiviral therapy but its poor
replication in vitro suggests that DNA polymerase mutations
will limit virulence in vivo.
Varicella-zoster virus glycoproteins
The VZV genome encodes seven known glycoproteins,
gB, gC, gE, gH, gI, gK and gL, as well as putative ORFs
for gM and gN. A number of recent publications have
addressed how VZV glycoproteins contribute to infectivity,
demonstrating how patterns of intracellular trafficking of
these proteins facilitate or are required for proper virion
assembly [915,16

]. VZV gB, like its herpesvirus

homologues, is highly conserved and probably has functions
that are essential for viral attachment and fusion to cell
membranes. Two regions within the gB cytoplasmic domain
have been shown to be required for transport from the
endoplasmic reticulum (ER) to the Golgi using transient
expression methods; these domains are a nine-amino-acid
motif (YMTLVSAAE) and the carboxy-terminal 17-amino-
acid segment [12].
VZV gE and gI are type 1 membrane proteins that form
heterodimers in infected cells. In contrast to gE, gI is
dispensable for replication in cell culture, but gI has chap-
erone functions that modulate gE trafficking and its
deletion is associated with lower virus yields and abnormal
syncytial formation. VZV gE and gI are encoded by ORFs
68 and 67, respectively, and are presumed to be expressed
primarily as late genes. RT-PCR experiments indicate that
gI and gE are transcribed both monocistronically and
bicistronically, with the monocistronic transcript being the
predominant species [14]. New evidence confirms the
importance of the gE and gI gene products as well as the
gE/gI complexes for VZVcell interactions. Further studies
of VZV-MSP have shown that the monoclonal antibody
escape mutation in the gE ectodomain confers a marked
enhancement in the capacity of the virus to spread from
cell to cell in cell culture and in skin implants in the
SCIDhu mouse model [2

]. In order to examine how gE

may interact with the host cell membranes that comprise
junctions between cells, Madin-Darby canine kidney
(MDCK) cells were constructed to express the glycoproteins
gE, gI, or gE/gI constitutively, and used to investigate the
effects of these VZV glycoproteins in polarized epithelial
cells [9]. VZV gE, expressed alone or with gI, induced
partial tight junction formation under low-calcium conditions
and colocalized with the tight junction protein, ZO-1.
Functionally, gE expression was accompanied by accelerated
establishment of maximum transepithelial electrical
resistance (which is a measure of resistance to transfer of
an electrical signal, used to demonstrate the formation of
physiologically functional tight junctions between cells) in
MDCKgE cells, and MDCKgI and MDCKgE/gI cells
exhibited a similar pattern, although peak resistances were
lower than those of gE alone. These studies indicate that
VZV gE and gE/gI may contribute to viral pathogenesis by
facilitating epithelial cellcell contacts, which could
enhance virus spread at mucosal sites.
VZV gE and gI undergo post-translational modifications in
the course of their maturation within infected cells and
extensive phosphorylation of gE has been demonstrated [17].
The phosphorylation of gI has now been analysed using a
fusion protein consisting of glutathione-S-transferase (GST)
and the gI cytoplasmic tail [11]. When the serine(343)
within a putative serine-proline phosphorylation site was
replaced with alanine, gI phosphorylation was reduced
significantly. Phosphorylation was mediated by the cellular
kinase, CDK1, which also phosphorylates gE, and by CDK2,
although not as effectively as CDK1 [11].
During VZV replication, gE and gI traffic to the surface of
infected cells, and also sort to the trans-Golgi network
(TGN), which appears to be a site of virion envelopment.
Whereas the gE endodomain has a TGN-signaling motif
that functions to retrieve gE from the cell membrane, gI
does not have equivalent motifs. Instead, TGN targeting
of gI expressed in Cos-7 cells has now been shown to
require the threonine(338) in the endodomain, as localization
was blocked if threonine(338) was deleted or changed to
alanine, serine, or aspartic acid [15]. The TIREE sequence,
which contains T(338), functions differently from those of
gE, which involve the retrieval of protein from the plasma
membrane. TIREE appears to be a TGN-retention signal.
Thus, both gE and gI have sequences that can function
independently to direct proteins to necessary sites on the
cell surface and intracellularly, but appropriate trafficking is
also enhanced by gE/gI complex formation. In experiments
done using partial or complete gI deletion mutants,
adjacent TGN cisternae were shown to adhere to one
another, resulting in marked distortions of the TGN by
electron microscopy. VZV virions did not reach post-Golgi
compartments when cells were infected with these gI
deletion mutants [16

].
Like gE and gI, gH and gL form heterodimers in infected
cells and these gH/gL complexes are likely to be involved
in cellcell fusion and virion egress. By analogy with other
herpesviruses, gH is probably essential for VZV replication.
In order to characterize gL more fully and to examine its
interactions with gH, these proteins were investigated
using vaccinia recombinants [13]. These experiments
showed that gL is made as 18 kDa and 19 kDa forms, with
443 Hostmicrobe interactions: viruses
the difference in size due to the addition of one endogly-
cosidase H-sensitive N-linked oligosaccharide, and that gL
appears to cycle between the ER and the cis-Golgi. When
gH and gL were expressed in the same cell, the 18 kDa
form of gL bound to mature gH (118 kDa), but the 19 kDa
gL did not make complexes with gH. In this system, gL
was required for maturation of gH from the precursor
97 kDa form to the 118 kDa form of the protein. Of note,
co-expression of gH and gL was associated with secretion
of gH/gL complexes, in which the gL form was 18 kDa
and gH was 114 kDa. VZV gE/gI complexes have not been
found to be secreted.
By sequence analysis, the VZV ORF5 encoded a putative
gK, a protein that is conserved among alpha-herpesviruses.
This protein has now been characterized as a 40 kDa
protein that is present in cell-free virus, and is detected in
virions and infected cell cytoplasm by immunogold electron
microscopy [10]. When gK deletion mutants were constructed
using VZV cosmids, complete and partial deletions of
ORF5 were incompatible with viral replication. Although
insertion of the HSV-1 gK gene into the endogenous VZV
gK site did not restore infectivity, placing the VZV gK gene
into a non-native AvrII site in the VZV genome yielded
infectious virus. When gK complementing cells were
transfected with the gK deleted cosmid and the three
intact cosmids, typical viral plaques were observed. These
observations indicate that gK is essential for VZV infectivity.
Varicella-zoster virus regulatory proteins
Several VZV gene products that are incorporated into the
virion tegument during assembly of mature virions have
transactivating functions for other viral genes. These proteins
are encoded by ORFs 4, 10, 61, and the duplicated genes,
62/71 and 63/70; ORF 61 also has transrepressor activity.
Several recent reports provide new information about VZV
regulatory proteins and their interactions [18,19

21

]. A
number of VZV genes encode proteins that have enzyme
activity, including the ORF47 and ORF66 gene products
which are serine/threonine protein kinases. Purification of
biologically functional VZV thymidine kinase expressed in
Escherichia coli has been accomplished and demonstrates
that this viral enzyme is active as a homodimer [22].
VZV IE62 protein is a predominant component of the virion
tegument and functions as the major immediate early
transactivator of VZV genes. IE62 protein also transctivates
some cellular promoters. This IE62 activation effect has
been analyzed in experiments showing that a TATA element
is necessary as well as sufficient [21

]. Of note, the efficiency

of IE62 promoter effects was different depending upon
variations of the TATA motif, which distinguished its
functional activity from the related HSV-1 protein, VP16.
These observations suggested that IE62 protein may
recognize conformational variations in DNA-bound
TATA-binding protein (TBP), TBP-transcription factor
IIA/B, or TBP-TATA-associated factor complexes [21

].
The IE62 protein has a potent nuclear localization signal in
infected cells and in expression systems. However, at late
times after infection, IE62 accumulates in the cytoplasm.
This phenomenon has been shown to depend upon phos-
phorylation of IE62 by the ORF66 serine/threonine
protein kinase [19

]. Co-expression of IE62 and ORF66

protein kinase reduced the capacity of IE62 to transactivate
reporter constructs, whereas ORF47 protein, the other VZV
kinase, had no effect. When an ORF66 stop codom mutant
was used to infect cells, IE62 retained its nuclear localization
throughout the cycle of infection. The active site of ORF66
required for its effects on intracellular IE62 localization
was mapped to its serine/threonine catalytic and ATP-
binding domains by mutational analysis and the essential
region of IE62 was amino acid residues 602 to 733 [19

].
The IE4 protein is known to function as a co-activator of
IE62-mediated transactivation in transient expression
experiments. The capacity of these two proteins to bind
was documented by co-immunoprecipitation and using
purified protein mixtures to show concentration dependent,
stable complex formation [20

]. The binding of IE4 was

enhanced with less-phosphorylated forms of IE62 protein
and could be mapped to a specific binding domain on IE62
protein [19

]. Since dimer formation is characteristic of

transcription factors, the formation of IE4 dimers and the
requirement of dimer formation for gene activation were
evaluated using a mammalian two-hybrid system [18].
These experiments showed that efficient dimer formation
depended upon residues in the central region and the
carboxy-terminal cysteine-rich domain (residues 443447)
of IE4 and that these domains must be intact for transac-
tivating function. Other domains, including the
arginine-rich domains Rb and Rc of the amino-terminal
region, also contributed to transactivation. IE4 was found
to have a nuclear localization signal in the Rb domain. With
respect to interaction with cellular factors, the optimal
nuclear export of IE4 was observed to require Crm-1, a
cellular export receptor also known as exportin 1.
In addition to viral transactivation, VZV gene expression
may be regulated by cellular factors. Analyses of the
intergenic regions of ORF 66/67 and ORF 67/68, containing
the putative promoters of gI and gE, using reporter constructs
indicate regions that are responsive to several cellular
transcription factors, including LAP/LIP, Sp1, YY1 and
NF-E2 [23]. Persistence of Sp1 mRNA and protein was
demonstrated at late times after VZV infection, suggesting
that synthesis of these host cell transcription factors may
not be blocked by viral replication [14].
Identification of ORF S/ L, a novel VZV gene
A new VZV ORF, designated ORF S/L, has been identified
recently at the left end of the VZV genome isomer; ORF
S/L has spliced mRNA transcripts that encode a series of
VZV proteins of 2130 kDa [24

]. Sequence comparisons
showed variability in ORF S/L among wild-type VZV isolates
and V-Oka, and protein products of different sizes were
detected in the cytoplasm when cells were infected with
Varicella-zoster virus Arvin 444
different VZV isolates. Disruption of the carboxy-terminal
region of ORF S/L altered the VZV phenotype by reducing
infected cell adherence and decreasing cellcell contact.
Expression of the ORF S/L protein was demonstrated
in human tissues infected with VZV but its function
remains unknown.
Immune evasion
In addition to its cytopathic effects, VZV infection has
more subtle effects on cellular protein expression, such as
interference with expression of gene productions involved
in immune clearance. VZV blocks the upregulation of
MHC class II expression, which is induced by interferon-
(IFN-), with inhibition occurring at the level of cellular
gene transcription through the JakStat pathway [25

].
Interference with MHC class II presentation of viral
peptides may reduce the initial CD4
+
T-cell response and
the lack of MHC class II expression on VZV-infected cells
from varicella and zoster lesions in vivo indicates that these
cells may be protected transiently from immune surveillance
by CD4
+
T cells. IFN- treatment induced cell surface
MHC class II expression on 60 to 86% of uninfected cells,
compared to 20 to 30% of cells that had been infected with
VZV prior to the addition of IFN-. In contrast, cells that
were treated with IFN- before VZV infection had MHC
class II expression similar to uninfected cells. In situ and
Northern blot hybridization of MHC II (MHC class II DR-a)
RNA expression in response to IFN- stimulation revealed
that MHC class II DR-a mRNA accumulated in uninfected
cells but not in cells infected with VZV. When skin biopsies
of varicella lesions were analyzed by in situ hybridization,
MHC class II transcripts were detected in areas around
lesions but not in cells that were infected with VZV. VZV
infection inhibited the expression of Stat 1 and Jak2
proteins but had little effect on Jak1. Analysis of regulatory
events in the IFN- signaling pathway showed that VZV
infection inhibited transcription of interferon regulatory
factor 1 and the MHC class II transactivator. This inhibition
of MHC class II expression on VZV-infected cells in vivo
may transiently protect cells from CD4
+
T cell immune
surveillance, facilitating local virus replication and trans-
mission during the first few days of cutaneous lesion
formation [25

].
VZV infection also reduces the usual constitutive MHC
class I molecules on the surface of infected cells [26

].
This effect has the potential to interfere with clearance of
VZV-infected cells by antigen-specific CD8
+
T cells. It is
observed on T cells infected with VZV, which may
facilitate viral transport to cutaneous sites as well as on
fibroblasts and may delay control of viral replication at
cutaneous sites. MHC class I molecules transit the ER but
are retained in the Golgi compartment of VZV-infected
cells, but the viral protein or proteins that mediate this
effect have not been identified. Flow cytometry showed
that VZV infection reduced the cell surface expression of
MHC class I on fibroblasts significantly, yet the expression
of transferrin receptor was not affected. Transferrin
receptor is a constitutively expressed protein that cycles to
the cell surface. The fact that its cell surface expression
was not disrupted indicated that the viral infection had not
disrupted all cellular proteins that have such a trafficking
pattern. Importantly, when human fetal thymus/liver
implants in SCID-hu mice were inoculated with VZV,
cell surface MHC class I expression was downregulated
specifically on VZV-infected human CD3
+
T lymphocytes,
a prominent target that sustains VZV viremia. The stage in
the MHC class I assembly process that was disrupted by
VZV in fibroblasts was examined in pulse-chase and
immunoprecipitation experiments in the presence of endo
H. MHC class I complexes continued to be assembled in
VZV infected cells, and were not retained in the ER. In
contrast, immunofluorescence and confocal microscopy
showed that VZV infection resulted in an accumulation of
MHC class I molecules that co-localized to the Golgi
compartment. Inhibition of late viral gene expression by
treatment of infected fibroblasts with phosphonoacetic
acid (PAA) did not influence the modulation of MHC
class I expression, nor did transfection of cells with plasmids
expressing immediate early viral proteins. However, cells
transfected with a plasmid encoding the early gene ORF66
did result in a significant downregulation of MHC class I
expression, suggesting that this gene product encodes an
immunomodulatory function. Thus, VZV downregulates
MHC class I expression by impairing the transport of
MHC class I molecules from the Golgi to the cell surface;
this effect may enable the virus to evade CD8
+
T-cell
immune recognition during VZV pathogenesis, including
the critical phase of T-lymphocyte-associated viremia [26

].
Cellular tropisms of VZV
The cell types that are involved in the pathogenesis of
VZV infection in the human host include T cells, skin cells
and neurons and satellite cells of the dorsal root ganglia.
New information about VZV tropism for differentiated
cells is difficult to acquire because of the highly species-
specific nature of VZV infection. In addition to clinical
studies, recent experiments demonstrate that VZV tropism
for some human cell types can be examined in the
SCIDhu mice that have allografts of human thymus/liver,
containing CD4
+
, CD8
+
and dual positive T cells, or human
skin implants that can be inoculated with VZV [2729].
Cell-associated viremia
VZV interactions with peripheral blood mononuclear cells
has been investigated in several recent publications
[26

,3035]. Using a quantitative PCR assay to detect

VZV DNA in peripheral blood, viral DNA was detected in
86% in patients with varicella and 81% of those with
herpes zoster; detection rates were 100 and 89%, respectively,
for samples obtained within the first week [32]. VZV could
be detected before antiviral treatment but not after acyclovir
was given to patients with acute varicella or herpes zoster
[31]. The highest viral load was present during varicella [35].
Although studies done with earlier methodssuggested that
chronic post-herpetic neuralgia (PHN) was associated with
445 Hostmicrobe interactions: viruses
persistent viremia, the use of standard and nested PCR
assays and isothermal transcription-based nucleic acid
amplification (NASBA) did not reveal the presence of VZV
DNA or RNA in peripheral blood mononuclear cells
(PBMCs) from patients with PHN [30].
In vitro methods to examine VZV T-cell tropism include
the infection of cord blood PBMCs [33] and II-23 cells, a
CD4-positive human T-cell hybridoma [34]. Using cord
blood, 34% of T cells were infected with VZV and mutants
that did not express ORF47 or ORF66 did not have normal
infectivity in T cells, in parallel with observations from the
SCIDhu-mouse model. V-Oka and low passage wild-type
VZV isolates were equally infectious for T cells but wild-
type VZV spread more efficiently to melanoma cells.
Infectivity of VZV for II-23 CD4
+
T cells was examined
using recombinant VZV expressing green fluorescent pro-
tein (VZV-GFP). II-23 cells were susceptible to VZV-GFP
infection as demonstrated by expression of immediate/early
(IE62), early (ORF4) and late (gE) genes but recovery of
infectious virus was limited, indicating that this system may
be most useful for evaluating viral entry into CD4
+
T cells.
Skin
When skin biopsy samples were examined for VZV gene
expression, immunohistochemical or in situ hybridization
methods revealed expression of IE63 in keratinocytes initially,
followed by gE, gB and IE63 synthesis in keratinocytes,
sebocytes, Langerhans cells, monocytes/macrophages and
endothelial cells; these VZV proteins were also detected in
dermal nerves and in perineural type I dendrocytes [36].
The ORF29 protein, the single-stranded DNA-binding
protein of VZV, a nonstructural nuclear protein, was found
in nerves of two of six patients with varicella. In conjunction
with cell culture observations, it was suggested that extra-
cellular ORF29 protein may be taken up human neurons
via endocytosis.
Neurotropism
Latent infection of cells in the dorsal root ganglia appears
to be an invariable consequence of primary VZV infection.
The viral determinants of virulence that are involved in
this process are not known and the cellular type or types
within which VZV persists has been uncertain. Transcripts
of ORFs 21, 29, 62, and 63 have been detected in autopsy
material and there is evidence that proteins of ORFs 4, 21,
29 and 62 are made in latently infected cells. Several recent
publications have revisited these issues using the latest
techniques to identify and quantify VZV [37,38

40

]. In
one series of 12 subjects, nine of whom had AIDS, dorsal
root ganglia were examined using in situ hybridization for
VZV RNA and DNA, and PCR in situ amplification for
DNA [37]. Sequences from distant segments of the
genome (ORFs 4 and 40) were detected in neurons from
ganglia of two normal and three AIDS cases, transcripts of
ORFs 4, 21, 29 and 63 were found in normal and AIDS
cases, and DNA and RNA corresponding to ORF29 was
detected in neurons in serial tissue sections in three cases
[37]. In further studies, these investigators examined
trigeminal ganglia (TG) from 35 subjects, 18 of whom had
HIV infection [38

]. ORF21 transcripts were detected in

7/11 normal and 6/10 HIV-positive subjects, ORF29
transcripts were detected in 5/14 normal and all of 11 HIV-
positive subjects, ORF62 transcripts were detected in 4/10
normal and 6/9 HIV-positive subjects, and ORF63 transcripts
were detected in 8/17 normal and 12/15 HIV-positive
subjects. ORF4 and ORF18 transcripts were detected in
some normal and HIV-positive subjects, whereas ORF28,
40, and 61 transcripts were rare or not detected in any
subjects. By immunohistochemical stain, IE63 protein was
expressed in TG cells of normal and HIV-positive subjects.
Explant cultures of TG, which harbor latent VZV, had a
broad range of VZV transcripts, compared to the restricted
pattern observed by direct analysis [38

]. The comparison
was between the detection of VZV genes in TG obtained
from immunocompetent individuals, and VZV genes in
TG obtained from individuals immunocompromised as a
result of HIV infection. Patients with HIV infection are at
risk of VZV reactivation from latency. Kennedy et al. wanted
to find out whether specific genes, or more VZV genes,
could be detected in the TGs of immunocompetent
individuals versus immunocompromised ones.
When copies of latent VZV in TG were assessed using real-
time fluorescence PCR, and compared with HSV-1 and
HSV-2 genome copies, 53% and 7% of 15 specimens were
positive for HSV-1 or HSV-2, with means of 2,902
+
1,082 SE
or 109 genomes/10
5
cells, respectively. From 7987% of 14
TG were positive for VZV ORFs 29, 31, or 62 [39

]. When
assays for all VZV genes were pooled, the mean was 258
+
38
VZV genomes/10
5
ganglion cells. Thus, VZV genome copy
numbers were substantially lower in TG compared with
HSV-1 genome copy numbers, which could help to account
for the high frequency of HSV-1 reactivation relative to VZV.
In another study using real-time PCR quantification to
determine the relative HSV and VZV DNA copy number in
TG from 17 subjects [40

], the number of HSV-1 genomes

ranged from 42.9677.9 copies per 100 ng of DNA and was
consistent between left and right TG from the same subject.
VZV genome copies ranged from 37.0 to 3,560.5 copies per
100 ng of DNA and were also consistent in left and right TG.
HSV-1 LAT transcripts were present in all HSV-1 PCR
positive TG. Of the three VZV transcripts analyzed, only
ORF63 transcripts were detected consistently [40

]. This
observation is consistent with prior reports that ORF63
expression is characteristic of VZV latency.
Memory T-cell immunity
Effective control of primary VZV infection and maintenance
of latency requires active immunity mediated by VZV-
specific T cells. The induction of these responses is likely
to depend upon antigen presentation by dendritic cells
(DC) [41]. Human DC were found to mediate in vitro
sensitization of naive CD4
+
T cells to synthetic peptides
corresponding to residues of VZV IE62 protein. When in vitro
responses to VZV peptide of naive T cells were compared
Varicella-zoster virus Arvin 446
with responses of T cells in vivo after immunization with
varicella vaccine, T-cell recognition was demonstrated for
three peptides in 71100% of the donors tested before and
after vaccination using DC as antigen-presenting cells. These
observations indicate that primary T-cell responses detected
in vitro using DC as APC may be useful to characterize
the potential immunogenicity of viral protein epitopes in vivo.
In the normal host with acute varicella, PBMCs functioned
to produce high concentrations of IFN-, tumor necrosis
factor- (TNF-), and interleukin (IL)-12 [42], consistent
with the predominant Th-1-like memory T-cell response
that is established after primary VZV infection.
As pathogens that persist after primary infection, control of
herpesviruses depends upon antigen-specific T-cell
responses but also requires sufficient numbers of memory
T cells. Frequencies of memory T cells specific for VZV,
HSV and human cytomegalovirus (HCMV) were compared
in immune adults by intracellular cytokine (ICC) detection
[43

]. The mean percentages of CD4

+
T cells were 0.11%
for VZV and 0.22% for HSV by IFN- production; the
frequency of CD4
+
T cells for HCMV was significantly
higher at 1.21%. Percentages of VZV

, HSV

and HCMV-
specific CD4
+
T cells were similar when TNF- was used
to detect antigen-specific responder cells. HCMV-stimulated
CD8
+
T cells produced IFN- (1.11%) and TNF- (1.71%);
however, VZV- and HSV-specific CD8
+
T cells were not
detectable using whole antigen as the stimulus. VZV CD4
+
T cell numbers were similar in young adults with natural
or vaccine-induced immunity but VZV CD4
+
T cells were
significantly less frequent in older adults, which is consistent
with their known susceptibility to VZV reactivation. These
results indicate that numbers of memory T cells specific
for particular herpesviruses may vary with the sites of viral
latency and with host age.
Clinical evidence of the significance of VZV specific T-cell
immune responses in hostvirus interactions is provided
by experience with the progressive nature of perinatal and
congenital infection; these infections occur in utero or in
early infancy when T-cell immune mechanisms are
deficient [4446]. In a series of 26 cases of neonatal varicella,
30% had dissemination and 26% had pneumonia [45]. In a
case of congenital varicella syndrome, malformations were
already apparent by sonography at 22 weeks; VZV DNA
was detected diffusely in fetal tissues and the placenta,
and was associated with miliary calcified necrosis in fetal
organs [44]. Adenosine deaminase deficiency is a genetic
disorder that results in impaired cell-mediated immunity;
when a child with undiagnosed adenosine deaminase
deficiency received varicella vaccine, the attenuated virus
caused hepatitis, further supporting the requirement of
effective adaptive T-cell immunity for control of VZV
replication even when the virus is attenuated [46].
VZV DNA vaccines
Three recent publications describe the use of DNA vaccines
to induce VZV-specific immunity in murine models [4749].
In an examination of the mechanism by which ORF62
(IE62 protein) and ORF68 (gE) plasmids induced immunity,
VZV proteins were shown to be expressed in non-regenerating
muscle cells. In response to primary immunization, muscle
cells did not express MHC class II transcripts and the
inflammatory response was limited, although some
humoral and cell-mediated responses were elicited. After a
second inoculation of ORF62-containing plasmid, protein
synthesis and a marked inflammatory infiltrate was evident.
PCR analyses demonstrated that ORF62 DNA in local
draining lymph nodes following primary DNA immunization
by intramuscular inoculation, indicating that transport of
plasmid DNA to sites of antigen presentation in regional
lymphoid tissue is important for initial generation of
immune responses; enhancement by secondary inoculation
followed trafficking of immune cells to the site of viral
protein synthesis in muscle cells [47]. When plasmids
expressing truncated gB or gE, lacking the anchor and the
cytoplasmic domains, were used to immunize mice, the gB
encoding plasmid was immunogenic but responses to gE
were limited [48]. However, a third report [49] confirmed
the initial study [47], showing that a gE-expressing
plasmid elicited VZV antibodies that were primarily IgG2a
subclass, consistent with induction of Th1-type immunity.
It is not clear that VZV DNA vaccines will be of major
clinical importance and efficacy cannot be evaluated in
mice, given the innate resistance to VZV infection, but some
information about the relatively immunogenicity of viral
proteins may be obtained.
Conclusions
Progress towards understanding the interactions between
VZV and its human host has been substantial, as newer
methods to examine viral gene functions have been devel-
oped, such as the generation of VZV mutants from cosmids
and confocal microscopy, flow cytometry and other methods
to examine interactions of single viral proteins with host
cells and effects on cellular gene expression, including
MHC class I and II molecules. The development of the
SCIDhu mouse model has permitted the evaluation of the
effects of targeted genetic mutations on VZV replication
during infection in vivo. Better tools for evaluating the
cellular tropisms of the virus, such as quantitative PCR,
have helped to define the role of T-cell viremia in patho-
genesis and to define patterns of gene expression during
latent infection of sensory ganglia. Finally, the licensure of
the live attenuated varicella vaccine has brought the study
of VZVhost interactions to a point at which it has had a
real impact upon the morbidity caused by this ubiquitous
human pathogen.
Update
New genetic analyses have shown that the ORF65 gene,
located in the short unique region of the genome, encodes a
small 16 kDa phosphorylated protein that localized to the Golgi
and was detected in virions [50]. ORF65 was dispensable
for replication in cell culture when the gene was deleted by
cosmid mutagenesis. With respect to virushost interactions,
447 Hostmicrobe interactions: viruses
granulysin, which is a lytic protein made by human natural
killer (NK) and cytotoxic T cells, has been found to have
direct antiviral activity against VZV-infected cells [51].
Granulysin enhanced death of VZV-infected cells by
apoptotic mechanisms and these effects were mediated by
the same amino acid residues, amino acids 2351, which are
responsible for the lysis of tumor cells by this protein.
Because granulysin is made by NK cells as well as cytotoxic
T lymphocytes (CTL), these observations indicate that
granulysin may play a role in innate, NK-mediated (as well
as adaptive, antigen-specific) host responses to VZV infection.
The Oka vaccine strain of varicella is known to have the
capacity to express foreign antigens. The Oka strain is
attenuated and safe for clinical use, suggesting that it may
be useful for vectored approaches to immunization against
pathogens other than VZV. Human immunodeficiency virus
(HIV) env antigen has now been expressed in this strain,
and induction of antibodies and delayed hypersensitivity
responses to the HIV antigen as well as VZV proteins was
demonstrated in guinea pigs [52]. Whether the Oka strain
expressing HIV env will be an effective vaccine is uncertain,
because single-protein vaccines are likely to have limited
protective potential, but it is likely that the VZV genome
will tolerate insertion of multiple foreign genes, including
those encoding other HIV proteins.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
of special interest
of outstanding interest
1. Vafai A, Forghani B, Kilpatrick D, Ling J, Shankar V: Stability of a
varicella-zoster virus glycoprotein E epitope. Arch Virol 2000,
145:85-97.
2. Santos RA, Hatfield CC, Cole NL, Padilla JA, Moffat JF, Arvin AM,
Ruyechan WT, Hay J, Grose C: Varicella-zoster virus gE escape
mutant VZV-MSP exhibits an accelerated cell-to-cell spread
phenotype in both infected cell cultures and SCI D-hu mice.
Virology 2000, 275:306-317.
The authors document that a single amino acid change in gE can confer
enhanced VZV virulence in vitro and in vivo.
3. Argaw T, Cohen JI, Klutch M, Lekstrom K, Yoshikawa T, Asano Y,
Krause PR: Nucleotide sequences that distinguish Oka vaccine
from parental Oka and other varicella-zoster virus isolates. J Infect
Dis 2000, 181:1153-1157.
4. Loparev VN, Argaw T, Krause PR, Takayama M, Schmid DS:
Improved identification and differentiation of varicella-zoster virus
(VZV) wild-type strains and an attenuated varicella vaccine strain
using a VZV open reading frame 62-based PCR. J Clin Microbiol
2000, 38:3156-3160.
5. Faga B, Maury W, Bruckner DA, Grose C: Identification and
mapping of single nucleotide polymorphisms in the varicella-
zoster virus genome. Virology 2001, 280:62-71.
6. Gomi Y, Imagawa T, Takahashi M, Yamanishi K: Oka varicella vaccine
is distinguishable from its parental virus in DNA sequence of
open reading frame 62 and its transactivation activity. J Med Virol
2000, 61:497-503.
The authors show that V-Oka has several changes in ORF62 that predict
altered amino acid residues; most importantly, they demonstrate that V-Oka
consists of subpopulations of VZV genomes that differ in sequence changes
from the parent Oka strain.
7. VZV vaccine (Merck & Yaminishi) and Parent Oka (Yaminishi) on
World Wide Web URL: http://cmgm.stanford.edu/~jjcheng/VZV/
8. Visse B, Huraux JM, Fillet AM: Point mutations in the
varicella-zoster virus DNA polymerase gene confers resistance to
foscarnet and slow growth phenotype. J Med Virol 1999, 59:84-90.
9. Mo C, Schneeberger EE, Arvin AM: Glycoprotein E of
varicella-zoster virus enhances cell-cell contact in polarized
epithelial cells. J Virol 2000, 74:11377-11387.
10. Mo C, Suen J, Sommer M, Arvin A: Characterization of
Varicella-Zoster virus glycoprotein K (open reading frame 5) and
its role in virus growth. J Virol 1999, 73:4197-4207.
11. Ye M, Duus KM, Peng J, Price DH, Grose C: Varicella-zoster virus Fc
receptor component gI is phosphorylated on its endodomain by a
cyclin-dependent kinase. J Virol 1999, 73:1320-1330.
12. Heineman TC, Krudwig N, Hall SL: Cytoplasmic domain signal
sequences that mediate transport of varicella-zoster virus gB from
the endoplasmic reticulum to the Golgi. J Virol 2000, 74:9421-9430.
13. Maresova L, Kutinova L, Ludvikova V, Zak R, Mares M, Nemeckova S:
Characterization of interaction of gH and gL glycoproteins of
varicella-zoster virus: their processing and trafficking. J Gen Virol
2000, 81:1545-1552.
14. Rahaus M, Wolff MH: Influence of different cellular
transcription factors on the regulation of varicella-zoster virus
glycoproteins E (gE) and I (gI) UTRs activity. Virus Res 1999,
62:77-88.
15. Wang ZH, Gershon MD, Lungu O, Zhu Z, Gershon AA:
Trafficking of varicella-zoster virus glycoprotein gI:
T(338)-dependent retention in the trans-Golgi network, secretion,
and mannose 6-phosphate-inhibitable uptake of the ectodomain.
J Virol 2000, 74:6600-6613.
16. Wang ZH, Gershon MD, Lungu O, Zhenglun Z, Mallory S, Arvin AM,
Gershon AA: Essential role played by the C-terminal domain of gI
in the envelopment of varicella zoster virus in the trans-Golgi
network: interactions of glycoproteins with tegument. J Virol 2001,
75:323-340.
The authors demonstrate remarkable changes in intracellular virion assembly
that occur when gI is deleted or expressed as a truncated protein.
17. Kinchington PR, Cohen JI: Viral proteins. In Varicella-Zoster Virus:
Virology and Clinical Practice. Edited by Arvin AM, Gershon AA.
Cambridge:Cambridge University Press; 2000.
18. Baudoux L, Defechereux P, Rentier B, Piette J: Gene activation by
varicella-zoster virus I E4 protein requires its dimerization and
involves both the arginine-rich sequence, the central part, and the
carboxyl-terminal cysteine-rich region. J Biol Chem 2000,
275:32822-32831.
19. Kinchington PR, Fite K, Turse SE: Nuclear accumulation of
I E62, the varicella-zoster virus (VZV) major transcriptional
regulatory protein, is inhibited by phosphorylation mediated by
the VZV open reading frame 66 protein kinase. J Virol 2000,
74:2265-2277.
This paper documents a novel role for the serine/threonine kinase encoded
by ORF66, a gene found only in the alpha-herpesviruses, in controlling viral
replication by its effects on IE62 phosphorylation.
20. Spengler ML, Ruyechan WT, Hay J: Physical interaction between
two varicella zoster virus gene regulatory proteins, I E4 and I E62.
Virology 2000, 272:375-381.
This paper provides the first evidence for direct physical binding of IE62, the
major transactivating protein of VZV, and IE4, suggesting that the ORF4 gene
product is important in VZV replication.
21. Perera LP: The TATA motif specifies the differential
activation of minimal promoters by varicella zoster virus
immediate-early regulatory protein I E62. J Biol Chem 2000,
275:487-496.
The author demonstrates significant differences between the mechanisms of
activation of viral and cellular genes by VZV IE62, compared to other her-
pesviral transactivators.
22. Amrhein I, Wurth C, Bohner T, Hofbauer R, Folkers G, Scapozza L:
Highly purified recombinant varicella Zoster virus thymidine
kinase is a homodimer. Protein Expres Purif 2000, 18:338-345.
23. Rahaus M, Wolff MH: Transcription factor Sp1 is involved in the
regulation of varicella-zoster virus glycoprotein E. Virus Res 2000,
69:69-81.
24. Kemble GW, Annunziato P, Lungu O, Winter RE, Cha TA,
Silverstein SJ, Spaete RR: Open reading frame S/ L of
varicella-zoster virus encodes a cytoplasmic protein expressed in
infected cells. J Virol 2000, 74:11311-11321.
The authors describe a previously unidentified ORF in VZV that spans the
S/ L segments and demonstrate that it encodes a protein product made in
cell culture and in vivo.
Varicella-zoster virus Arvin 448
25. Abendroth A, Slobedman B, Lee E, Mellins E, Wallace M, Arvin AM:
Modulation of major histocompatibility class I I protein expression
by varicella-zoster virus. J Virol 2000, 74:1900-1907.
This paper demonstrates that VZV blocks upregulation of MHC class II in
response to IFN- and that the effect is via disruption of the signal for gene
transcription via the Jak/Stat pathway.
26. Abendroth A, Lin I, Slobedman B, Ploegh H, Arvin AM: Varicella
zoster virus retains major histocompatibility complex class I
proteins in the Golgi of infected cells. J Virol 2001, 75:4878-4888.
The authors report that VZV downregulates MHC class I, not only in cell cul-
ture but on infected human T cells. This effect occurs by a pathway not pre-
viously described for herpesviral interference with MHC class I surface
expression (i.e. Golgi retention) and a novel class of viral protein, the ORF66
viral kinase, is implicated in modulation of MHC class I expression.
27. Moffat JF, Stein MD, Kaneshima H, Arvin AM: Tropism of varicella-
zoster virus for human CD4
+
and CD8
+
T-lymphocytes and
epidermal cells in SCI D-hu mice. J Virol 1995, 69:5236-5242.
28. Moffat J, Zerboni L, Stein M, Grose C, Kaneshima H, Arvin A: The
attenuation of the vaccine Oka strain of varicella-zoster virus and
the role of glycoprotein C in alphaherpesvirus virulence
demonstrated in the SCI D-hu mouse. J Virol 1998, 72:965-974.
29. Moffat J, Zerboni L, Sommer MH, Heineman TC, Cohen J, KaneshimaH,
Arvin A: The ORF47 and ORF66 putative protein kinases of varicella-
zoster virus determine tropism for human T cells and skin in the
SCI D-hu mouse. Proc Natl Acad Sci USA 1998, 95:11969-11974.
30. Schunemann S, Mainka C, Wolff MH: No acute varicella-zoster
virus replication in peripheral blood mononuclear cells during
postherpetic neuralgia. Acta Virol 1999, 43:337-340.
31. Wolff MH, Schunemann S: Acyclovir treatment prevents
varicella-zoster virus replication in PBMC during viremia. New
Microbiol 1999, 22:309-314.
32. de Jong MD, Weel JF, Schuurman T, Wertheim-van Dillen PM, Boom R:
Quantitation of varicella-zoster virus DNA in whole blood, plasma,
and serum by PCR and electrochemiluminescence. J Clin
Microbiol 2000, 38:2568-2573.
33. Soong W, Schultz JC, Patera AC, Sommer MH, Cohen JI: Infection of
human Tlymphocytes with varicella-zoster virus: an analysis with
viral mutants and clinical isolates. J Virol 2000, 74:1864-1870.
34. Zerboni L, Sommer M, Ware CF, Arvin AM: Varicella-zoster virus
infection of a human CD4-positive T-cell line. Virology 2000,
270:278-285.
35. Kimura H, Kido S, Ozaki T, Tanaka N, Ito Y, Williams RK, Morishima T:
Comparison of quantitations of viral load in varicella and zoster.
J Clin Microbiol 2000, 38:2447-2449.
36. Annunziato PW, Lungu O, Panagiotidis C, Zhang JH, Silvers DN,
Gershon AA, Silverstein SJ: Varicella-zoster virus proteins in skin
lesions: implications for a novel role of ORF29p in chickenpox.
J Virol 2000, 74:2005-2010.
37. Kennedy PG, Grinfeld E, Gow JW: Latent Varicella-zoster virus in
human dorsal root ganglia. Virology 1999, 258:451-454.
38. Kennedy PG, Grinfeld E, Bell JE: Varicella-zoster virus gene
expression in latently infected and explanted human ganglia.
J Virol 2000, 74:11893-11898.
See annotation [40

].
39. Pevenstein SR, Williams RK, McChesney D, Mont EK, Smialek JE,
Straus SE: Quantitation of latent varicella-zoster virus and herpes
simplex virus genomes in human trigeminal ganglia. J Virol 1999,
73:10514-10518.
See annotation [40

].
40. Cohrs RJ, Randall J, Smith J, Gilden DH, Dabrowski C, van Der Keyl H,
Tal-Singer R: Analysis of individual human trigeminal ganglia for
latent herpes simplex virus type 1 and varicella-zoster virus
nucleic acids using real-time PCR. J Virol 2000, 74:11464-11471.
This paper, along with Kennedy et al. [38

] and Pevenstein et al. [39

], adds
substantially to information about VZV latency in human tissues, showing the
quantification of viral genome copies in ganglia and the difference between
viral gene transcripts that are found in latency versus those that are identified
after explant culture.
41. Jenkins DE, Yasukawa LL, Bergen R, Benike C, Engleman EG, Arvin
AM: Comparison of primary sensitization of naive human T-cells
to varicella-zoster virus peptides by dendritic cells in vitro with
responses elicited in vivo by varicella vaccination. J Immunol 1999,
162:560-567.
42. Torigo S, Ihara T, Kamiya H: I L-12, I FN-gamma, and TNF-alpha
released from mononuclear cells inhibit the spread of
varicella-zoster virus at an early stage of varicella. Microbiol
Immunol 2000, 44:1027-1031.
43. Asanuma H, Sharp M, Maecker HT, Maino VC, Arvin AM:
Frequencies of memory T-cells specific for varicella-zoster virus,
herpes simplex virus, and cytomegalovirus by intracellular
detection of cytokine expression. J Infect Dis 2000, 181:859-866.
This paper demonstrates that the antigen-specific responder cell frequencies
within the CD4
+
T-cell subpopulation is significantly lower in healthy individuals
latently infected with VZV or HSV, alpha-herpesvirues that are latent in neuronal
cells, versus human cytomegalovirus, a beta-herpes virus that persists in cells
of the immune system.
44. Hartung GE, Chaoui R, Arents A, Tennstedt C, Bollmann R: Prenatal
diagnosis of congenital varicella syndrome and detection of
varicella-zoster virus in the fetus: a case report. Prenat Diagn
1999, 19:163-166.
45. Singalavanija S, Limpongsanurak W, Horpoapan S, Ratrisawadi V:
Neonatal varicella: a report of 26 cases. J Med Assoc Thai 1999,
82:957-962.
46. Ghaffar F, Carrick K, Rogers BB, Margraf LR, Krisher K, Ramilo O:
Disseminated infection with varicella-zoster virus vaccine strain
presenting as hepatitis in a child with adenosine deaminase
deficiency. Pediatr Infect Dis J 2000, 19:764-766.
47. Abendroth A, Slobedman B, Springer ML, Blau HM, Arvin AM:
Analysis of immune responses to varicella zoster viral proteins
induced by DNA vaccination. Antiviral Res 1999, 44:179-192.
48. Massaer M, Haumont M, Garcia L, Mazzu L, Bollen A, Jacobs P,
Jacquet A: Differential neutralizing antibody responses to
varicella-zoster virus glycoproteins B and E following naked DNA
immunization. Viral Immunol 1999, 12:227-236.
49. Hasan UA, Harper DR, Argent S, Layton G, Wren BW, Morrow WJ:
Immunization with a DNA expression vector encoding the
varicella zoster virus glycoprotein E (gE) gene via intramuscular
and subcutaneous routes. Vaccine 2000, 18:1506-1514.
50. Cohen JI, Sato H, Srinivas S, Lekstrom K: Varicella-zoster virus
(VZV) ORF65 virion protein is dispensable for replication in cell
culture and is phosphorylated by casein kinase I I, but not by the
VZV protein kinases. Virology 2001, 260:62-71.
51. Hata A, Zerboni L, Sommer M, Kaspar AA, Clayberger C, Krensky AM,
Arvin AM: Granulysin blocks replication of varicella-zoster virus and
triggers apoptosis of infected cells. Viral Immunol 2001, 14:225-243.
52. Shiraki K, Sato H, Yoshida Y, Yamamura J, Tsurita M, Kurokawa M,
Kageyama S: Construction of Oka varicella vaccine expressing
human immunodeficiency virus env antigen. J Med Virol 2001,
64:65-69.
449 Hostmicrobe interactions: viruses