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Journal of Virology, December 2001, p. 11573-11582, Vol. 75, No. 23
Centro de Biología Molecular
"Severo Ochoa" (Universidad Autónoma de Madrid-Consejo
Superior de Investigaciones Científicas), 28049 Cantoblanco,
Madrid, Spain
Received 21 August 2001/Accepted 25 August 2001
The infection outcome of the Parvoviridae largely
relies on poorly characterized intracellular factors modulated by
proliferation, differentiation, and transformation of host cells. We
have studied the interactions displayed by the highly homologous p and
i strains of the murine parvovirus minute virus of mice (MVM), with a
series of transformed cells of rat (C6) and human (U373, U87, SW1088, SK-N-SH) nervous system origin, seeking for molecular mechanisms governing parvovirus host range. The MVMp infection of C6 and U373
cells was cytotoxic and productive, whereas the other nervous cells
behaved essentially as resistant to this virus. In contrast, MVMi did
not complete its life cycle in any of the human nervous cells, though
it efficiently killed the astrocytic tumor cells by two types of
nonproductive infections: (i) normal synthesis of all viral
macromolecules with a late defect in infectious virion maturation and
release to the medium in U373; and (ii) high levels of accumulation of
the full set of viral messenger RNAs and of both nonstructural (NS-1)
and structural (VP-1 and VP-2) proteins, under a very low viral DNA
amplification, in U87 and SW1088 cells. Further analyses showed that
U87 was permissive for nuclear transport of MVMi proteins, leading to
efficient assembly of empty viral capsids with a normal phosphorylation
and VP1-to-VP2 ratio. The DNA amplification blockade in U87 occurred
after conversion of the incoming MVMi genome to the monomeric
replicative form, and it operated independently of the delivery pathway
used by the viral particle, since it could not be overcome by
transfection with cloned infectious viral DNA. Significantly, a
chimeric MVMi virus harboring the coding region of the nonstructural
(NS) gene replaced with that of MVMp showed a similar pattern of
restriction in U87 cells as the parental MVMi virus, and it attained in
U373 cultures an infectious titer above 100-fold higher under equal levels of DNA amplification and genome encapsidation. The results suggest that the activity of complexes formed by the NS polypeptides and recruited cellular factors restrict parvovirus DNA amplification in
a cell type-dependent manner and that NS functions may in addition determine MVM host range acting at postencapsidation steps of viral
maturation. These data are relevant for understanding the increased
multiplication of autonomous parvovirus in some transformed cells and
the transduction efficacy of nonreplicative parvoviral vectors, as well
as a general remark on the mechanisms by which NS genes may regulate
viral tropism and pathogenesis.
Viral infection begins with the
specific recognition of cell surface receptors by virus structural
components followed by additional interactions leading to irreversible
internalization of the particle. In order to be productive, the
infection must successfully proceed through several processes, such as
uncoating and genome expression, replication, and maturation. The
family Parvoviridae, a large group of small viruses
containing a linear single-stranded (ss) DNA genome of approximately
5,000 nucleotides (23, 77), constitutes an
interesting and useful system to dissect molecular mechanisms of
virus-host interactions due to the genetic simplicity of its members
and the multiplicity of cellular functions on which parvovirus
multiplication relies. Indeed, in addition to the cellular surface
molecules acting as specific receptors for some members of the three
genera of the family, as for the adeno-associated virus (AAV)
(68, 81, 82), the human erythrovirus B19
(11), and the autonomous parvovirus Aleutian Disease Virus
(ADV) (38) and Canine Parvovirus (CPV) (64),
the multiplication of parvoviruses requires functions expressed during
the S phase of the cell cycle (5, 83, 87) as well as
factors expressed at certain differentiation (55, 80, 84)
and transformation (56, 86) stages, the nature of which
remains largely unknown.
The genome of the autonomously replicating members of the parvovirus
genus is organized into two overlapping transcription units that are
timely regulated (18). The left gene, driven by the P4
promoter, encodes the NS1 and NS2 nonstructural proteins, and the right
gene, driven by the P38 promoter, encodes the VP1 and VP2 structural
proteins. Sixty VP protein subunits assemble to form the parvoviral
capsid, which structure at atomic resolution has been resolved for the
canine parvovirus (CPV) (89) and the immunosuppressive
strain of the minute virus of mice (MVMi) (1), among
others. The two NS polypeptides play diverse roles in virus multiplication. The smaller NS2 protein (28 kDa) contains three isoforms arising from alternate splicings (26) that can
bind the cell cycle regulator 14-3-3 protein family (10)
and shuttle from the nucleus to the cytoplasm via the CRM1 export
pathway (8). A multiplicity of functions has been assigned
to NS2, such as capsid assembly (29), messenger
translation (59), and DNA replication and virus production
in a cell type-specific manner (48, 58), though the NS2
mode of action remains unclear. The larger NS1, the main viral
replicator protein (23), is a multifunctional nuclear
phosphoprotein (19) that performs crucial steps of the
MVM's unique rolling-hairpin mode of DNA synthesis (reviewed in
reference 28). Viral DNA replication starts with the
conversion reaction, the so-called synthesis of the complementary strand of the ss virion genome by cellular factors (5)
generating a double-stranded (ds) monomer replicative form (mRF) DNA.
The DNA binding, nicking, and helicase activities of NS1 (27,
60), together with the concourse of additional cellular factors
(15-17), are strictly required for the accomplishment of
the subsequent replication steps, including the introduction of an ss
nick, the amplification of mRF DNA to multimeric intermediates, and the resolution and packaging of virus genomes (27, 72).
Moreover, NS1 is a potent transactivator of the viral promoters
(32) by the recognition of DNA elements (51,
71) and mediates cytotoxic effects that may be modulated by the
cell physiological stage (14).
Determinants of parvovirus tropism have been identified in different
regions of the genome. Sequences within the NS gene were found to be
important for the extension of murine parvovirus MVM (44)
and porcine parvovirus (PPV) (90) host ranges, although the mechanism of action was not further explored. More data are available for the cell tropism determinants laying in the capsid gene,
demonstrated for MVM (2, 44), CPV (65), PPV
(6), and ADV (7, 39). Two strains of MVM, the
prototype (MVMp) and the immunosuppressive (MVMi) strains isolated from
cell cultures (9, 30), which differ in just a few amino
acids of their coding sequences (3) but show
characteristic tropism to mouse lymphohemopoietic cells in vitro
(35, 54) and distinct pathogenicity in newborn mice
(12, 47, 69) and SCID mice (76), were used to
delineate tropism determinants for the infection of fibroblast and
lymphoid cell lines in vitro (85). The main capsid
determinant for MVM productive infection (2, 44), the
so-called allotropic determinant, was mapped to two amino acids of the
threefold spike of the capsid which, functioning coordinately
(4) via the VP-2 protein (53), mediates the
interaction between the incoming particle (43) and
intracellular factors (78). In the nonfunctional conformation of this determinant, the MVM infection is restricted prior
to transcription and gene expression (2, 43) by a proposed altered decapsidation after nuclear transport of the virions
(67) that delays the initiation of viral DNA synthesis and
prevents DNA amplification (67, 78).
The complexity of the cellular factors involved in parvovirus
multiplication is best illustrated by the privileged interaction of
these viruses with transformed cells (reviewed in reference 73). In the genus of the autonomous parvovirus, this
phenomenon, called parvoviral oncosuppression, has been mainly
evaluated for the H-1 and MVMp viruses infecting human and mouse
fibroblasts and epithelial cells transformed by a variety of agents
that lower intracellular restrictions to parvovirus multiplication
(21, 56), and in which some oncogenes were particularly
efficient for this effect (57, 74). The facilitated
multiplication of parvoviruses in transformed cells (or oncotropism)
was in correspondence with an increased gene expression and
transcription activity from the P4 promoter (20, 22). In
addition, some neoplastic cells seemed to be particularly sensitive to
the cytotoxicity of parvovirus NS proteins (14, 57),
accounting for their favored killing by these viruses under in vitro
(86) and in vivo (34, 88) experimental conditions.
The potential use of parvoviruses as anticancer biological reagents
demands a comprehensive understanding of their tropism toward
transformed cells. For this purpose, we have explored a system formed
by the homologous MVMp and MVMi strains interacting with a collection
of reference cell lines derived from rat and human central nervous
system tumors of neural and glial origin, at graded malignant stages.
Cytopathic effect (CPE) of the cultures, virus growth, cytotoxicity,
and viral macromolecular biosynthesis were monitored for each
virus-cell interaction. Thereby, we found two distinct nonproductive
MVMi infections internally restricted in human astrocytic tumor cells,
in which either viral full gene expression and empty capsids production
proceeded in the absence of DNA amplification or the DNA-full virions
produced to normal levels showed a low specific infectivity. The
features of these infections may underlie novel molecular mechanisms of
parvovirus host range determination by nonstructural functions.
Cells.
Cells isolated from the nervous system were purchased
from the American Type Culture Collection. The U87 MG human
glioblastoma (HTB-14), U373 MG human glioblastoma (HTB-17), SW1088
human astrocytoma (HTB-12), SK-N-SH human neuroblastoma (HTB-11), and
C6 rat glioma (CCL-107) cells were cultured in Dulbecco's modified
Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf
serum (FCS), except that SW1088 cells were cultured in DMEM without sodium bicarbonate in closed tissue-culture flasks with 30 mM HEPES
buffer. All the cells were maintained for a minimal number of passages.
Viruses.
Purified stocks of the immunosuppressive strain of
the parvovirus MVM (MVMi) and of the prototype strain (MVMp) were
prepared from their respective infectious molecular clones, pMVMi and
pMM984 (44). The viral plasmids, amplified in
Escherichia coli strain JC8111 cells that preserve the viral
hairpins and enriched in supercoiled forms by chromatography
(Qiagen), were electroporated into the cells in which these viruses
were originally isolated, the EL-4 mouse C57BL T-cell lymphoma
(9) and the A9 mouse fibroblast (30) cell
lines, respectively. Cells were cultured for 48 h, and the
intracellular virus was harvested and used to infect at a low
multiplicity of infection (MOI) the same permissive cell lines. Large
viral stocks devoid of empty capsids were subsequently prepared by
density gradient equilibrium centrifugation and stored at
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11573-11582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genome Replication and Postencapsidation Functions
Mapping to the Nonstructural Gene Restrict the Host Range of a Murine
Parvovirus in Human Cells
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ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C
(76). Infectious virus was quantified by plaque assay on
monolayers of the NB324K simian virus 40-transformed human newborn
kidney cells as previously described (85).
Measurement of cell viability. The effect of MVM infection on the viability of the cell lines was quantified by a clonogenic assay based in a previously described method (20). Cells seeded at a density of 5,000/cm2 were infected at increasing MOI (0.2 to 10 PFU/cell), and at 4 h postinoculation (hpi), cells were trypsinized and plated at various densities (2 × 102 to 104 cells per 60-mm-diameter dish) in triplicate. Cells were incubated for 10 days in the proper medium supplemented with a neutralizing dilution of MVM capsid antiserum to block reinfections, and arising colonies were fixed in absolute methanol and stained with 1% crystal violet. Survival is expressed as the percentage of colonies in the infected cultures with respect to the uninfected cultures normalized for a similar number of plated cells.
Blot analyses of MVM nucleic acids. For viral transcription analysis, total RNA of infected cells was prepared as previously described (70), electrophoresed in agarose gels with 6% formaldehyde, and blotted onto nylon membranes (Gene Screen Plus; Dupont) by capillary transfer. The amount of loaded RNA was controlled by visualizing in the membranes the ribosomal RNAs stained with methylene blue. For Southern analysis of virus DNA replication, infected or transfected cells were extensively washed with phosphate-buffered saline (PBS) and processed for low-molecular-weight DNA extraction by a modified Hirt procedure (54) with carrier tRNA to ensure quantitative recoveries. DNA was fractionated by agarose gel electrophoresis and alkali blotted onto nylon membranes. Both types of membrane-bound samples were hybridized to a gel-purified MVM full-length DNA probe 32P labeled by random priming. When indicated, a 32P-labeled negative-sense riboprobe was synthesized with T7 bacteriophage RNA polymerase by using a PstI (nt 2129)-NcoI (nt 3121) restriction fragment of the MVMp genome cloned in a pGEM vector as previously described (70). Hybridizations were carried out at 42°C overnight in 50% deionized formamide, 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5× Denhardt's solution, 10% dextran sulfate (Pharmacia), 0.5% sodium dodecyl sulfate (SDS), and 200 µg of denatured salmon sperm DNA per ml. Membranes were washed at 56°C in 0.1× SSC-0.5% SDS and exposed for autoradiography to Kodak X-Omat films for the indicated time periods.
Viral protein synthesis and subcellular distribution. Cell lines seeded at 10,000 cells/cm2 were infected at an MOI of 5 PFU/cell with MVMp or MVMi and labeled 10 to 24 hpi in methionine-free DMEM supplemented with 10% normal medium, 10% dialyzed FCS, and 100 µCi of [35S]Met-Cys (Amersham AGQ 0080)/ml. At the end of the labeling period, cells were washed in PBS and disrupted in 50 mM Tris (pH 8.0)-0.15 M NaCl-0.2% SDS and a mixture of protease inhibitors by flushing through a 25-gauge needle. The level of synthesis of the VP1 and VP2 capsid proteins and of the NS1 nonstructural protein in the cell extracts was determined by immunoprecipitation with specific antibodies raised against NS1 and MVM capsid in rabbits (75), and the immunocomplexes were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) analysis. The subcellular localization of the MVM capsid and NS1 proteins in the infected cells was determined with the same antibodies by indirect immunofluorescence (IF) as previously described (50).
Transfection.
Cells were transfected by the conventional
calcium phosphate method as previously described (70). In
brief, 10 µg of supercoiled plasmid was spotted as precipitated on
monolayers of 106 cells growing in a
90-mm-diameter petri dish. After overnight incubation, cells were
extensively washed with PBS and samples were taken either immediately
(time zero posttransfection) or 48 h afterwards. To monitor that
the MVMi plasmid was internalized into the nucleus of the NB324K and
U87 cells, parallel cultures were similarly transfected with the
addition of 2 µg of a reporter plasmid expressing the E. coli 
-galactosidase enzyme under control of the MVM P4 promoter
(to be described elsewhere) and stained accordingly 48 h posttransfection.
Analysis of MVM particle formation. Infected cellular monolayers (106 cells) 35S labeled 10 to 24 hpi as above were washed and scrapped in PBS, 0.2% SDS was added, and DNA was sheared by flushing through a 25-gauge needle. The homogenates were brought to 9 ml in PBS-0.2% SDS and centrifuged for 18 h at 16,000 rpm and 15°C in a Beckman SW40 rotor (30,000 × g) through a 3-ml sucrose cushion (20% sucrose, 50 mM Tris [pH 8.0], 0.1 M NaCl, 1 mM EDTA, 0.2% SDS) to select for viral proteins assembled in particles. Pellets were resuspended and brought to 10 ml of 20 mM Tris (pH 8.0)-1 mM EDTA-0.2% Sarcosyl, adjusted to a density of 1.38 g/ml in CsCl by refractometry (ni = 1.370), and centrifuged to equilibrium for 42 h at 48,000 rpm and 15°C in a Beckman Ty65 rotor (150,000 × g). Gradients were fractionated from the top, and the positions of the 35S label-empty MVM capsids (1.32 g/ml) and DNA-full virions (1.41 to 1.46 g/ml) were determined by scintillation counting. Our method to prepare purified 32P-labeled MVM empty capsids and to perform two-dimensional analysis of VP2 tryptic phosphopeptides has been recently described (52).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Distinct types of MVM infections in transformed cells of nervous
system origin.
As a preliminary test of permissiveness, growing
cultures of human (U373, U87, SW1088, and SK-N-SH) and rat (C6)
transformed cells of the nervous system were inoculated at a low
MOI (0.1 PFU/cell) with the parvovirus strains MVMp and MVMi,
and the progression of the CPE arising in the cultures, manifested by
detached rounded cells and a lower density of the monolayers, was
inspected daily under microscope (Fig.
1A). The NB324K cell line used as a
productive system to grow these viruses was the only cell type highly
susceptible to both strains. MVMp caused extensive CPE in C6 and U373
cultures and undetectable CPE in U87, SW1088, and SK-N-SH cultures (not shown). MVMi instead caused at this low MOI a moderate CPE in C6
cultures, but it failed to produce any CPE in the assayed human cell
cultures. However, when the infections were performed at a high MOI (5 PFU/cell), the extent of the CPE at 3 days postinoculation caused by
each of these viruses further varied, since MVMi was able to cause a
pronounced CPE in the three human astrocytic tumor cells (U87, U373,
and SW1088), whereas the CPE of the MVMp infections remained unchanged
(not shown). These results were consistent in several independent
inoculations.
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Uneven macromolecular biosynthesis of parvovirus MVM in nervous
tumor cells.
To investigate the molecular bases of the distinct
MVM infections, we performed a quantitative analysis of the
biosynthesis of viral macromolecular components in a single round of
infection. Viral DNA synthesis analyzed by Southern blotting showed
early in the virus-cell interactions (2 hpi) a similar amount of
single-stranded input viral genome associated to the entire set of cell
lines used (Fig. 2), an indication that
virions of both MVM strains can attach to them. At a later time (20 hpi), only the NB324K, C6, and U373 cells denoted permissiveness for a
high level of synthesis of MVMp and MVMi DNA replicative intermediates
and genomic forms (Fig. 2). This result was in agreement with the
productive character of these cells both for MVMp and, with the
exception of U373, for MVMi as well (Fig. 1B). However, MVMi failed to
amplify DNA to easily detectable levels in any of the other human cell lines (Fig. 2, right), in spite of the fact that it efficiently killed
the U87 and SW1088 astrocytic tumor cells (Fig. 1C).
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Virus particle formation in nonproductive MVMi infections.
The
apparent lack of infectious MVMi production in the medium of the human
glioblastoma cultures prompted us to investigate the fate of the
abundant viral proteins synthesized in these cells. To assess the
capacity of the structural proteins to assemble in viral particles,
35S-labeled protein extracts of MVMi infected
U373 and U87 cells (MOI of 5) were subjected to equilibrium
centrifugation in CsCl gradients. An intracellular accumulation of
empty (
= 1.32 g/ml) and DNA-full ( = 1.41 g/ml) particles
at the normal rate found in MVM-infected cells (54) was
obtained in U373 extracts (not shown), in agreement with the
quantitative ssDNA production demonstrable in blots (Fig. 2) that is
believed to result from viral genome encapsidation (23).
As depicted in Fig. 4A, the U87 cells
yielded also high levels of particles sedimenting at the position of
empty capsids; however, no significant peak of
35S counts was detected at the banding position
of the DNA-full virion. These empty particles showed all the features
of the MVM capsids recovered from productively infected NB324K cells,
namely hemagglutination activity of mouse erythrocytes, a complex
pattern of phosphorylation demonstrable by two-dimensional tryptic
analysis (52), and a protein composition of VP1 to VP2 at
an approximate ratio of one to five (data not shown). The VP antigen
accumulated in the nucleus of the infected U87 cells (Fig. 4B), the
subcellular compartment where the MVMi assembly occurs
(50). NS1, the major viral replicative polypeptide of MVM
reported to translocate into the nucleus of murine and human permissive
cells (22, 23), also accumulated normally into the nucleus
of most U87 cells of the cultures (Fig. 4C). The intense and general
nuclear staining indicated that most U87-infected cells expressed the
structural and the NS1 cytototoxic protein, corresponding with the high
proportion of these cells killed by the MVMi infection in clonogenic
assays (Fig. 1C, left). These data showed that none of the evaluated protein properties, including level of synthesis, nuclear transport and
capsid assembly, accounts for the abortive character of the MVMi
infection in U87 cells.
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MVMi replication in U87 glioblastoma is blocked after DNA
conversion step.
The apparent lack of MVMi DNA amplification in
U87 (Fig. 2) was inconsistent with the high level of viral gene
expression reached in these cells (Fig. 3), given that MVM
transcription initiation requires the conversion of the incoming ss
viral genome to a dsDNA template (5), allowing the onset
of P4 promoter activity. To get insights into the mechanisms
restricting MVMi DNA amplification in U87 cells, the synthesis of the
viral intermediate replicative forms in transfected and infected U87
cells was carefully examined. The MVMi infectious plasmid clone was
efficiently replicated in transfected control permissive NB324K cells,
as the viral ss genomes and the replicative intermediates (mRF and dRF)
accumulated by 48 h posttransfection (Fig.
5A). In contrast, none of these viral DNA
molecules was resolved in transfected U87 cells, and the signal of the
plasmid weakened with time (Fig. 5A).
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NS gene of MVMp enhances MVMi multiplication in U373 human
glioblastoma.
The features of MVMi abortive infection in U373 and
U87 cells drew our interest to the viral nonstructural (NS1 and NS2)
proteins, as known mediators of many replicative and maturation
functions during MVM infection. The U373 cells, in which the MVMp fully productive cycle (Fig. 1 to 3) ensures functional NS proteins and
proper collaboration of cellular factors, provided a system in which a
hypothetical involvement of NS proteins in MVMi abortive infections
could be tested. For this purpose, an intertypic recombinant virus
carrying most of the NS1 and NS2 coding sequence (NSp) of MVMp cloned
into the MVMi genome background (MVMi-NSp) was constructed, grown in
and purified from NB324K cells, and used for comparison of its DNA
replication and maturation capacities in glioblastoma cells infected at
different MOI with those of the parental MVMi. This recombinant virus
showed, very much like MVMp, a larger plaque size in NB324K cells (not
shown). At an MOI of 0.1, both viruses synthesized by 24 hpi abundant
DNA replicative intermediates (mRF and dRF) and virus genomes (ss) in
U373 cells (Fig. 6A, lane 2). In U87
cells, however (Fig. 6A, lanes 3 and 4), no significant viral DNA
synthesis was detected besides the conversion of the input ss genomes
into mRF forms. Similarly, at an MOI of 5, the synthesis of replicative
intermediates and of ss forms corresponding to encapsidated genomes
occurred to comparably high levels for both viruses only in U373 cells
(Fig. 6A, right). Thus, the activities endowed by the NS coding
sequence of MVMp (NSp) maintained the level of MVMi DNA replication in
U373 but did not overcome the restrictions imposed by the U87 cells.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Intracellular host range restrictions to parvovirus infections. This report adresses a comprehensive assessment of the interaction of the parvovirus MVMp and MVMi strains with transformed cells of nervous system origin. The characteristics of the infections were remarkably diverse within the series of cells tested (Fig. 1). Indeed, both viruses completed a cytotoxic and productive cycle exclusively in the rat glioma C6 cells. In the human cells, the astrocytoma U373 was the only nervous cell line fully permissive to MVMp, and the rest behaved as resistant and nonproductive hosts for this viral strain. MVMi did not complete a productive cycle in any of the human nervous cells, but its interaction with the three human tumor astrocytic cells (U373, U87, and SW1088) led to highly cytotoxic infections demonstrable in clonogenic assays (Fig. 1C, right) noncorresponded with a significant production of infectious virus in the culture media (Fig. 1B, right). The consistent evidence on distinct types of infection caused by each strain of this murine parvovirus in the human transformed nervous cells prompted us to further analyze this system in search of novel mechanisms of parvovirus host range determination.
At the molecular level, the MVMi abortive infections of the astrocytic tumor cells were not restricted at the same step of virus life cycle. In U373 cells, the virus macromolecular synthesis was complete and occurred to high levels (Fig. 2 and 3), but the production of mature infectious virions was low (Fig. 6B) and the exit from the cells to the medium very inefficient (Fig. 1B, right), hampering the progression of the infection in the culture (Fig. 6C). In the MVMi cytotoxic and abortive interaction with the human U87 and SW1088 cells, the block of the virus cycle was at an earlier step. These cells tolerated high levels of viral transcription and protein synthesis but no significant DNA amplification (Fig. 2 and 3), as the conversion reaction of the input viral genome to mRF could only be detected in blots hybridized with high-specific-activity probes (Fig. 5B and 6A). The NS1 and the structural viral proteins translocated and accumulated efficiently in the nucleus in the cellular population, forming empty capsids (Fig. 4) with a normal VP1-to-VP2 ratio. Even in the U87 cultures infected at a low MOI (Fig. 6C), most cells showed at a late postinfection time a characteristic-punctuated cytoplasmic staining for MVMi structural proteins, presumably due to the capacity of the empty capsids produced in high amounts by the few initially infected cells to reenter uninfected ones. Thus, all the analyses focused on capsid synthesis, composition, and properties suggest that it is the nonavailability of multimeric replicative DNA forms to be encapsidated what hampers virions formation in the U87 cells. The main features of the MVMi life cycle in the human astrocytic tumor cells set these abortive infections apart from previous reports on parvovirus host range determination, in which the virus macromolecular synthesis was also investigated (2, 43, 63, 65, 90). Particularly in MVM, the best characterized system, the MVMp and MVMi nonproductive infections of mouse fibroblast and lymphoid cells are restricted prior DNA amplification, and neither transcription nor gene expression are detectable (2, 43, 78). The viral determinant allowing a productive interaction with fibroblasts, named the allotropic determinant (44), was mapped within a region of the capsid gene that acts with the incoming particle, as the restriction could be overcome by DNA transfection (43). Similar allotropic-like determinants mapping to the VP gene and operating by a few adjacent residues have been described for the PPV, ADV, and CPV parvoviruses (6, 7, 65, 66, 90). Capsid determinants may also underlie the lack of MVMp transcription and gene expression in U87 cells (Fig. 3) since the MVMi-NSp virus, harboring the NS gene of MVMp packaged into an MVMi coat, killed U87 cells (not shown) and expressed capsid proteins in the first round of infection (Fig. 6C). In contrast, although the abortive MVMi infection of the human U87 and SW1088 cells reported here was also restricted before DNA amplification (Fig. 2), viral transcription and gene expression proceeded normally (Fig. 3), and the restriction could not be overcome by transfection with an infectious molecular clone (Fig. 5A). Thus, in these human transformed nervous cells, the MVMi infection is restricted at the intracellular level by mechanisms in which the virus particle is not involved. The unbalanced macromolecular biosynthesis in the abortive MVMi infection of U87 cells may shed light into the course of the viral life cycle along a nonrestricted infection. Indeed, the data indicate that high levels of transcription and gene expression may be reached by using as template the limited number of mRF molecules resulting from the conversion reaction. A similar phenomenon may occur in a normal productive infection, while the destiny of the dRF and higher concatemer replicative intermediates resulting from the virus DNA amplification might be the packaging into maturing capsids. This hypothesis is supported by the data obtained from dependovirus AAV vectors in which transduction was correlated with the conversion of the vector genome to dsDNA forms (36), as well as from the lack of correlation between gene expression and viral DNA amplification in MVM-based vectors (33).Mechanisms of host range control by NS functions. The intertypic recombinant MVMi-NSp virus amplified DNA and yielded ss genomic forms in the astrocytic U373 cells to the same high level as the parental MVMi (Fig. 6A), although the infectious titers attained by the virus (Fig. 6B) and the progression in U373 cultures infected at a low MOI (Fig. 6C) were significantly higher. As the ss genomes result from the encapsidation process (23), the yield of virus particles in U373 is the same in both viruses, and thus the specific infectivity of the newly formed MVMi-NSp virions must be severalfold higher. Therefore, the NSp gene enables an efficient production of infectious virus in U373 cells acting at as-yet-unidentified postencapsidation steps of the MVMi maturation process. A contribution of NS functions to the ordered genome packaging within the MVMi coat (1) may increase virion stability or uncoating in the subsequent infection. The copy of the NS1 protein placed outside the infectious virion covalently linked to the encapsidated 5' end of the ss MVM genome (25), as a possible factor mediating the quality of the packaging steps, is a tentative hypothesis to be investigated.
On the contrary, the restrictions imposed by the human U87 and SW1088 astrocytic tumor cells to MVMi DNA synthesis could not be overcome by the NSp gene (Fig. 6), and no infectious virus was produced. Here, the mRF DNA of MVMi serves as adequate template for the transcription machinery (Fig. 3A), but the NS functions necessary for DNA amplification seemed to be hampered. NS1 as well as NS2 have been described to be necessary for viral DNA amplification and efficient virus production, although the NS2 activities were dispensable in human transformed cells (48, 58) and necessary for efficient messenger translation (48, 59), a defect not seen in U87 or SW1088 cells (Fig. 3). NS1, however, is necessary in any cell type for the replication steps following the conversion reaction that leads to DNA amplification (27, 60, 72), and thus it is the main candidate polypeptide for explaining the lack of MVMi genome amplification in the tumor astrocytic cells. What is particularly intriguing is why the NS1 protein of MVMi expressed to high levels in U87 and SW1088 cells and normally translocated to the nucleus failed to amplify DNA, even though the other well-established NS1 mediated functions, such as P38 promoter transactivation (32) or cytotoxicity, which indeed can be modulated in transformed cells (14, 57), were not affected (Fig. 1 to 4). Interestingly enough, NS1 replicative functions can be modulated in vitro by phosphorylation through members of the protein kinase C family (31, 61), and some of these phosphorylations may also take place in the viral infection (19). We are presently investigating whether the NS1 phosphorylation state differ between transformed cells. Alternatively, a number of proteins have been described to assist on NS1 functions (15-17, 24) which may be altered or expressed to lower levels in certain neoplastic cells. Given the few coding differences between the NS proteins of the MVM strains (3), the human tumor astrocytic cells provide an useful system to map those residues functionally involved in the different NS1 activities, as well as to work out components of the replication machinery of transformed cells recruited for parvovirus replication. It is worth questioning whether the effects that the NS functions exert on MVM host range may be an exclusive phenomenon of the particular system of transformed nervous cells used in this report and are thus of poor biological interest for other viral-host interactions in the Parvoviridae. There are, however, previous genetic evidences for an involvement of NS sequences in parvovirus host range reported for PPV and MVM restricted infections (44, 90). In addition, MVMp may kill transformed fibroblasts independently of virus production (46), with gene expression uncoupled from DNA replication in some types of transformed fibroblasts (37). In the erythrovirus genus, the B19 parvovirus suppresses primary human megakaryocyte colony formation by a nonproductive interaction in which a low level of viral transcription was supported without DNA replication (79) and in nonpermissive cell lines blockades in viral transcript maturation (49) or capsid protein production (41) have been described. Furthermore, our results may provide a support of basic knowledge for the increasing evidences of the important roles that NS genes play on host range and tropism in diverse viral systems in vivo (e.g., references 13 and 62). In polyomavirus, in which the NS genes play host range functions as well (40, 42), the distribution of the initially targeted lung cells of inoculated mice showing viral DNA synthesis and proteins expression did not match (45). A dependence for NS functions and intracellular factors expressed at certain differentiation stages that restrict viral genome amplification may be a general phenomenon governing the tropism of the small DNA viruses.| |
ACKNOWLEDGMENTS |
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M.-P. Rubio and S. Guerra contributed equally to this work.
We are indebted to P. Tattersall for kindly providing the infectious molecular clones of MVMp and MVMi viruses and to J. Rommelaere and J. Cornelis for helpful comments.
This work was supported by grant SAF 98-0019 from the Comisión Interministerial de Ciencia y Tecnología and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular "Severo Ochoa." M.-P.R. was supported by a contract and S.G. was supported by a predoctoral fellowship, both from the Spanish Ministry of Education.
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FOOTNOTES |
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* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain. Phone: 34-913978048. Fax: 34-913978087. E-mail: jmalmendral{at}cbm.uam.es.
Present address: Instituto de Biomedicina de Valencia (CSIC), 46010 Valencia, Spain.
Present address: Centro Nacional de Biotecnología (CSIC),
28049 Cantoblanco, Madrid, Spain.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, December 2001, p. 11573-11582, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11573-11582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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