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Herpes
simplex virus selectively induces expression of the CC chemokine
RANTES/CCL5 in macrophages through a mechanism dependent on PKR and ICP0.
J.
Virol. (2002) 76:2780-2788.
Melchjorsen J, Pedersen FS,
Mogensen
SC
,
Paludan SR.
Department of Medical Microbiology and Immunology, University of Aarhus,
Denmark.
Recruitment of leukocytes is essential for eventual control of virus
infections. Macrophages represent a leukocyte population involved in the
first line of defense against many infections, including herpes simplex
virus (HSV) infection. Through presentation of antigens to T cells and
production of cytokines and chemokines, macrophages also constitute an
important link between the innate and adaptive immune systems. Here, we
have investigated the chemokine expression profile of macrophages after
HSV infection and the virus-cell interactions involved. By reverse
transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and
HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine
macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13
was also induced in peritoneal cells. Twenty-six other chemokines tested
were not affected. Accumulation of RANTES mRNA was detectable after 5 h of
infection, was sensitive to UV irradiation of the virus, and was preceded
by accumulation of viral immediate-early mRNA and proteins. The viral
components responsible for initiation of RANTES expression were examined
with virus mutants and RAW 264.7 macrophage-like cells expressing a
dominant negative mutant of the double-stranded-RNA-activated protein
kinase (PKR). The PKR mutant cell line displayed reduced constitutive and
HSV-inducible RANTES expression compared to the control cell line. HSV-1
mutants deficient in genes encoding the immediate-early proteins ICP4,
ICP22, and ICP27 remained fully capable of inducing RANTES expression in
macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to
induce RANTES expression was compromised. Thus, HSV selectively induces
expression of RANTES in macrophages through a mechanism dependent on
cellular PKR and viral ICP0.
Parvovirus
Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a
Reconstituted DNA Replication System.
J Virol. 2002 Jul;76(13):6518-31.
Christensen J,
Tattersall P.
Institute of Medical Microbiology and Immunology,
Panum Institute, University of Copenhagen, Copenhagen 2200 N, Denmark.
Departments of Laboratory Medicine and Genetics, Yale University School of
Medicine, New Haven, Connecticut 06510.
We show here that the DNA helicase activity of the parvoviral initiator
protein NS1 is highly directional, binding to the single strand at a
recessed 5' end and displacing the other strand while progressing in a
3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA
binding factor, PIF, also known as glucocorticoid modulating element binding
protein, bind to the left-end minimal replication origin of minute virus of
mice, forming a ternary complex. In this complex, NS1 is activated to nick
one DNA strand, becoming covalently attached to the 5' end of the nick in
the process and providing a 3' OH for priming DNA synthesis. In this
situation, the helicase activity of NS1 did not displace the nicked strand,
but the origin duplex was distorted by the NS1-PIF complex, as assayed by
its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides
on both strands of the nicked origin underwent limited unwinding. Addition
of Escherichia coli single-stranded DNA binding protein (SSB) did not lead
to further unwinding. However, addition of recombinant human single-stranded
DNA binding protein (RPA) to the initiation reaction catalyzed extensive
unwinding of the nicked origin, suggesting that RPA may be required to form
a functional replication fork. Accordingly, the unwinding mediated by NS1
and RPA promoted processive leading-strand synthesis catalyzed by
recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal
left-end origin cloned in a plasmid as a template. The requirement for RPA,
rather than SSB, in the unwinding reaction indicated that specific NS1-RPA
protein interactions were formed. NS1 was tested by enzyme-linked
immunosorbent assay for binding to two- or three-subunit RPA complexes
expressed from recombinant baculoviruses. NS1 efficiently bound each of the
baculovirus-expressed complexes, indicating that the small subunit of RPA is
not involved in specific NS1 binding. No NS1 interactions were observed with
E. coli SSB or other proteins included as controls.
Detection of orientation-specific anti-gp120
antibodies by a new N-glycanase protection assay.
APMIS 2002 Feb;110(2):123-31
Gram GJ, Bolmstedt A, Schonning K, Biller M, Hansen J-ES,
Olofsson S.
Department of Clinical Virology, University of
Goteborg, Sweden.
Several functions have been assigned to the extensive
glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape
mechanisms, but the intramolecular interactions between gp120 and its
carbohydrate complement are not well understood. To analyse this phenomenon
we established a new microwell deglycosylation assay for determining
N-linked glycan accessibility after binding of gp120-specific agents.
Orientation-specific exposition of gp120 in ELISA microplates was achieved
by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205.
We found that soluble CD4 inhibited the deglycosylation of gp120 only when
gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from
HIV-infected individuals inhibited the deglycosylation best when gp120 was
caught by NEA9205. These results demonstrated that both the CD4-binding site
and the epitopes recognised by antibodies from HIV-infected individuals have
N-glycans in the close vicinity. However, the difference in gp120
orientation indicates that antibodies in HIV-infected individuals, at least
partly, bind to epitopes different from the CD4-binding site. Finally, we
determined the structural class of the glycan of one V1 glycosylation site
of prototype HIV-1 LAI gp120, which remained unsolved from previous studies,
and found that it belonged to the complex type of glycans.
Embryonic stem cell production through
therapeutic cloning has fewer ethical problems than stem cell harvest from
surplus IVF embryos.
J Med Ethics 2002 Apr;28(2):86-8
Hansen J-ES.
Centre for Rare Diseases and Disabilities,
Copenhagen, Denmark. jesh@dadlnet.dk
Restrictions on research on therapeutic cloning are
questionable as they inhibit the development of a technique which holds
promise for successful application of pluripotent stem cells in clinical
treatment of severe diseases. It is argued in this article that the ethical
concerns are less problematic using therapeutic cloning compared with using
fertilised eggs as the source for stem cells. The moral status of an
enucleated egg cell transplanted with a somatic cell nucleus is found to be
more clearly not equivalent to that of a human being. Based on ethical
considerations alone, research into therapeutic cloning should be encouraged
in order to develop therapeutic applications of stem cells.
Baculovirus
expression of erythrovirus V9 capsids and screening by ELISA: serologic
cross-reactivity with erythrovirus B19. J Med Virol. 2002
Feb; 66:246-52
Heegaard E.D., Qvortrup K. and J. Christensen.
Department of Clinical Microbiology, University
State Hospital, Rigshospitalet, Denmark. e.heegaard@immi.ku.dk
Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of
viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly
different from erythrovirus B19 (> 11% nucleotide disparity), was
isolated. Standard B19 PCR assays were inconclusive and serologic tests
failed to categorize V9 as an acute B19-like infection. Sequencing, combined
with PCR studies, have since demonstrated the need for specific and
differentiated techniques when examining samples for possible B19 or V9
viremia. The antigenic properties of the V9 capsid proteins have not been
characterized previously. To address this question, V9 VP1 and VP2 open
reading frames were cloned and expressed in insect cells using a baculovirus
vector. Large quantities of purified recombinant V9 capsid protein were
produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and
VP2 capsids into empty icosahedral erythrovirus-like particles with a
diameter of approximately 23 nm. Screening of a panel of 270 clinical
samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100%
serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids
to a commercial B19 VP2 assay. This suggests that both a V9 and a B19
antibody response may be diagnosed equally well by ELISA using either V9 or
B19 recombinant capsids as antigen source. Retrospectively, translation of
the V9 sequence indicates that despite a significant genetic variation on
the DNA level, the majority of the discrepant DNA sequence represents silent
mutations leading to an amino acid sequence very similar to the known B19
strains (96-97% homology).
Detection
of Parvovirus B19 NS1-Specific Antibodies by ELISA and Western blotting
Employing Recombinant NS1 Protein as Antigen. J
Med Virol. 2002
July, 67:375-83
Erik D.
Heegaard , Cecilie J. Rasksen and Jesper
Christensen.
Department
of Clinical Microbiology ,University State Hospital, Rigshospitalet, and
Institute of Medical Microbiology and Immunology, University of Copenhagen,
Copenhagen, Denmark.
Human parvovirus B19 (B19) encodes a
number of non-structural proteins including the major protein NS1, and two
structural proteins VP1 and VP2. Utilization of denatured NS1 in ELISA and
Western blot (WB) assays have provided an opportunity to study some of the
immunologic properties of NS1, but results have been equivocal and the
diagnostic sensitivity poor probably because of the absence of
conformational epitopes. Presently, various viral isolates and baculovirus
vectors were employed to produce recombinant B19 NS1 under non-denaturing
conditions for the first time. To assess the antigenicity of purified B19
NS1, the reaction pattern of 252 samples were compared by B19 NS1 and VP2
ELISA. In sera from individuals with past infection (VP2 IgG positive),
using this new antigen significantly increased the sensitivity of ELISA
compared to WB (78% versus 33%, P=0.001), thereby contradicting perpetuated
claims that B19 NS1 IgG is primarily detected in patients with arthralgia or
chronic infection. Previous reports of NS1 IgG being absent during the
initial phase of infection (<
6 weeks) were proved incorrect by the detection of NS1 IgG in 60% of samples
from patients recently infected by B19. Applying conformational epitopes in
ELISA consequently raises the diagnostic sensitivity, although
immunologically, a temporal (years) attenuation of NS1 antibodies appears to
take place. This novel diagnostic tool may be useful as a supplement in case
of borderline results by VP2 ELISA and in monitoring the efficacy of future
capsid-based B19 vaccines.
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