Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK1
Centre National de la Recherche Scientifique, Laboratoire de Genetique des Virus, 91198 Gif-sur-Yvette Cedex, France2
Author for correspondence: Tony Minson.Fax +44 1223 336 926. e-mail acm{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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Our knowledge of the receptors with which these proteins interact remains superficial. Cell surface heparin proteoglycan is required for infection (WuDunn & Spear, 1989 ) and it is supposed that the interaction of the heparin-binding protein gC with cell surface glycosaminoglycans constitutes an important first step in infection. However, gB also binds heparin (Herold et al., 1994
) and this interaction may be of importance in adsorption or penetration. A new member of the immunoglobulin superfamily and a poliovirus receptor homologue have been identified as receptors for gD (Whitbeck et al., 1997
; Geraghty et al., 1998
). Receptors for the gH:L complex have not been identified.
It is uncertain whether the infection of sensory neurones occurs by the same processes as infection of fibroblasts or epithelial cells. HSV-1 mutants lacking glycoproteins E, I, J or G absorb to and penetrate fibroblasts and epithelial cells normally but, in mouse infections, exhibit reduced neurovirulence to varying degrees (Balan et al., 1994 ; Griffiths et al., 1998
). It is impossible to know whether these attenuated phenotypes reflect a specific deficiency for neuronal infection, but more detailed studies indicate that the gE and gI of HSV-1 and pseudorabies virus play an important role in neuroinvasion and, in particular, that mutants lacking these proteins fail to infect second order neurones (Card et al., 1992
; Card & Enquist, 1995
; Dingwell et al., 1995
; Babic et al., 1996
). These findings raise the possibility that infection of neurones by HSV-1 might require the participation of viral glycoproteins different from, or in addition to, those required for the infection of fibroblasts or epithelial cells. The objective of the work described here was to examine the ability of a series of HSV-1 mutants, each lacking an individual glycoprotein, to infect primary sensory neurones in culture.
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Methods |
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We failed to construct a gB-negative LacZ+ virus on a background of HSV-1 SC16 and resorted to using a mutant based on the HFEM strain. This was constructed using a genomic KpnI fragment (nucleotide 5273757438) and inserting the lacZ expression cassette into the SnaB1 site at nucleotide 54269, in the central region of the gB coding sequence. This plasmid was co-transfected with HSV-1 HFEM DNA into the gB helper cell line D6 (Cai et al., 1988 ) and a LacZ+ recombinant virus was isolated. The resulting isolate, HFEM-
UL27-Z, was helper cell-dependent and produced no detectable gB in Vero cells infected at an m.o.i. of 3.
HSV-1 SC16-C3b contains the same lacZ expression cassette used in all other mutants inserted within the LAT locus (Lachmann & Efstathiou, 1997 ). The growth of this virus in vitro and during acute infection in vivo is indistinguishable from the parent (S. Efstathiou, personal communication). SC16-C3b was used as a ß-galactosidase-positive surrogate for wild-type virus in most experiments.
All cells were grown in Glasgow modified Eagle's medium supplemented with 10% bovine foetal serum. Wild-type virus and replication competent mutants were propagated and assayed on BHK-21 cells. The gH-negative and gB-negative mutants were grown on the helper cell lines CR1 and D6 respectively (Cai et al., 1988 ; Boursnell et al., 1997
). The gD-negative mutant was isolated using the VD60 helper cell line (Ligas & Johnson, 1988
), but propagation of the mutant on these cells resulted in the generation of contaminating gD+LacZ- progeny, presumably due to recombination. A gD helper cell line was, therefore, constructed which contained no gD flanking sequences in common with the mutant. The gD promoter and coding sequence (nucleotides 138019139606) was isolated as an EcoRV fragment as described above and ligated to the CMV IE polyadenylation sequence (+2757 to +3053 with respect to the IE transcript start) derived from plasmid MV10. This cassette was inserted into plasmid pSP73 (Promega) and was co-transfected into Vero cells with plasmid pcDNA-3 (Invitrogen). Transfected cells were selected by growth in G418 and individual colonies were tested for their ability to support the growth of a gD-negative virus. A clone of cells was identified and named Vero gD+/19.
Production of virions lacking gB, gD or gH was achieved by infection of BHK cells with the relevant mutant at an m.o.i. of 3. The progeny viruses were quantified by particle counting using the electron microscope.
Preparation of primary rat sensory neurones.
Newborn (day 13) Wistar rats were killed by decapitation and the entire spinal column dissected to yield 3040 dorsal root ganglia from levels C1 to L6. The ganglia were suspended in Ham's F14 medium containing 4% Ultroser-G (Gibco) and treated with 200 µg/ml collagenase types IV and XI (Sigma) for 2 h at 37 °C. The cells were mechanically dissociated by pipetting and the suspension centrifuged at 200 g for 15 min. The pellet was washed twice in Ham's medium and centrifuged through 15% BSA in Ham's medium to remove debris. The cells were then preplated twice for 15 min on plates coated with 0·5 mg/ml type VII rat tail collagen to reduce the non-neuronal cell population.
Cultures were established on 13 mm glass coverslips which were pre-treated for 1 h with 0·5 mg/ml poly-dl-ornithine in water and for 1 h with 10 µg/ml laminin in Ham's F14 medium. Each coverslip received 1000 cells in 100 µl Ham's F14 medium containing 4% Ultroser-G, 2 mM glutamine, 1·25 µg/ml penicillin and streptomycin, 20 ng/ml nerve growth factor (2·5S mouse NGF, Boehringer) and 50 µM 5-fluoro-5'deoxyuridine to prevent growth of mitotic cells. After 3 h a further 200 µl of the same medium was added. Cultures were used after 3 or 4 days after neurite outgrowth had occurred.
Histochemical detection.
Cultures were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and cells were permeabilized with 0·1% Triton X-100 in PBS at room temperature for 5 min. The cultures were then blocked by incubation for 30 min in 10% horse serum and 15% BSA in PBS. Neurones were identified by staining with mouse anti-human ß-tubulin type III (Sigma) followed by Cy3-conjugated donkey anti-mouse IgG or by staining with rabbit anti-mouse neurofilament 200 (Sigma) followed by Cy2-conjugated goat anti-rabbit IgG. Infected cells were detected by staining with a mixture of monoclonal antibodies LP2 and AP2, against gD and VP5 respectively (McLean et al., 1982 ). Cells infected with viruses expressing ß-galactosidase were detected by staining with X-Gal.
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Results |
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To establish that mutant SC16-C3b could be used as a LacZ+ surrogate for wild-type virus, parallel cultures were infected with various doses of strain SC16 or SC16-C3b. After 7 h cultures infected with SC16 were stained for virus antigens and cultures infected with SC16-C3b were stained for virus antigens or ß-galactosidase. Neurones were identified by staining for neurofilament 200 (in cultures stained for virus antigens) or ß-tubulin (in cultures stained for ß-galactosidase). The proportion of neurones infected was then determined in each culture. Table 1 shows the combined results of two experiments and establishes that SC16-C3b and the parent virus, SC16, infect neurones with equal efficiency. In all subsequent experiments SC16-C3b was used as a surrogate for wild-type virus.
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Infection with virions lacking dispensable glycoproteins
Virions lacking glycoproteins C, E, G, I or J are infectious and previous studies with strain SC16 mutants lacking these glycoproteins showed that their particle to infectivity ratios were indistinguishable from that of the wild-type parent, whether infectivity was measured on BHK cells or on polarized epithelial cells (Balan et al., 1994 ; Griffiths et al., 1998
). To determine whether any of the proteins play a significant role in the infection of neurones, cultures were infected with increasing doses of each mutant or with SC16-C3b, and after 7 h the cultures were stained for ß-galactosidase expression and for ß-tubulin. In these experiments the `input' inoculum is based on p.f.u. measured in BHK cells, but since the particle to infectivity ratio (as measured on BHK cells) of each mutant is similar, the inocula contain equivalent numbers of virions. Table 2
gives the outcome of an experiment in which multiple cultures were infected with each mutant and the proportion of neurones staining positive for ß-galactosidase was determined 7 h after infection. The results show that replicate cultures give reasonably reproducible results and that virions lacking gE, gG, gI or gJ infect neurones with similar efficiency to SC16-C3b. Virions lacking gC infected neurones with somewhat reduced efficiency. It is difficult to place a value on this reduction in efficiency, but it appears that five to ten times as many gC-negative virions are required to achieve a comparable proportion of infected neurones. Infection of neurones by gC-negative viruses and SC16-C3b was compared in a second set of cultures, but the outcome was scored 13 h after infection. Table 3
shows that similar results were obtained. To verify that this phenotype was due to the engineered mutation and not to a fortuitous mutation elsewhere in the genome a second independent gC-negative mutant was constructed using the same method (SC16-
UL44-ZB). This mutant was then used to infect further cultures and also exhibited a reduced ability to infect neurones (Table 4
). We thought it possible that the culture conditions, rather than neurone susceptibility, might be responsible for the reduced infectivity of gC-negative virions. For example, gC-negative virions might absorb more efficiently to the poly-dl-ornithine/laminin-coated coverslips. The cultures used to obtain the data in Table 4
were therefore re-examined and scored for the proportion of ß-galactosidase-positive non-neuronal cells. The results (Table 5
) were ambiguous. Firstly, the proportion of infected cells in relation to virus dose is not consistent with a Poissonian distribution, implying that all cells are not equally infectible. This is hardly surprising given the mixture of cell types likely to be present. Secondly, the gC-negative virions appear to infect slightly less efficiently than SC16-C3b virions, though the difference is not as great as that observed when infection of neurones was scored.
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Discussion |
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We observed that two independent mutants lacking gC infected neurones with a lower efficiency (of about five- to tenfold) than wild-type virus, whereas these mutants have specific infectivities which are indistinguishable from wild-type on BHK, Vero and epithelial cells (Griffiths et al., 1998 ). We regard this reduced efficiency as marginal, particularly since the same mutants exhibited a slightly lower efficiency of infection of non-neuronal cells in the same culture. Nevertheless, our results support those reported recently by Immergluck et al. (1998)
who found that gC-negative mutants exhibited an entry defect on primary chick neurones.
It is worth noting that our results do not rule out a role for gG, gE, gI or gJ in infection of neurones in vivo. In these circumstances viruses must enter sensory nerve endings from epithelium at the site of infection, whereas, in culture, the entire neuronal body and neurite surface is available for infection. Nevertheless, the results reported here, together with the reported phenotype of the mutants in mice (Balan et al., 1994 ), argue against a role for these glycoproteins in infection of first order neurones.
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Acknowledgments |
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Footnotes |
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c Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.
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References |
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Received 21 April 1999;
accepted 26 May 1999.