1 School of Biology and Biochemistry, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, UK
2 Molecular Pathology Laboratory, Royal Group of Hospitals Trust, Belfast BT12 6BL, Northern Ireland, UK
3 School of Medicine, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, UK
4 Molecular Medicine Program, Mayo Clinic, Guggenheim 18, Rochester, MN 55905, USA
Correspondence
W. Paul Duprex
p.duprex{at}qub.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Various types of persistently infected cell cultures have been generated and characterized. Some are so called carrier cultures' and these represent a dynamic equilibrium between replication of the virus and division of the host cell. Uninfected cells are present in these cultures and treatment with antiviral antiserum can cure the cultures from the infection. These carrier cultures are often susceptible to crises in which most cells die due to extensive virus replication. Both interferons and defective interfering particles have been implicated in controlling the equilibrium (Collins & Flanagan, 1977). Other persistently infected cell cultures, which are probably more relevant to persistence in tissues, are characterized by infection of most, if not all of the cells. These divide normally and a constant level of virus replication and gene expression is observed. In some cases infectious virus is released. Several cell lines of this type have been generated with MV and related morbilliviruses using a number of strategies (Burnstein et al., 1974
; Joseph et al., 1975
; Rustigan, 1966
). These true persistently infected cells are typified by differing degrees of superinfection immunity. This extends to infection by related morbilliviruses but not to other paramyxoviruses or interferon-sensitive viruses from other families, indicating that cytokines are not primary controlling factors (Fernandez-Muñoz & Celma, 1992
; Rager-Zisman et al., 1984
). It has always been a challenge to determine the extent of superinfection immunity, as assessment has relied on directly observing distinct cytopathic effects or indirectly detecting viral antigen.
Recently, recombinant viruses expressing fluorescent proteins have been used to examine all stages of the virus life cycle (Duprex et al., 1999b; Elliott & O'Hare, 1999
; Husain & Moss, 2001
; Oomens & Wertz, 2004
). Studies in plants, transgenic mice and zebrafish (Niwa et al., 1999
; Moss et al., 1996
; Villuendas et al., 2001
) have reported no detrimental effects to these organisms upon green fluorescent protein (GFP) expression and a recombinant canine distemper virus (CDV) expressing enhanced green fluorescent protein (EGFP) retains its virulence in animals (von Messling et al., 2004
). Thus, apart from very isolated examples (Huang et al., 2000
; Liu et al., 1999
), GFP is not considered to be cytotoxic. Thus, we reasoned that viruses expressing fluorescent proteins could be used effectively for the study of viral persistence. The emission and fluorophore maturation properties of the fluorescent protein DsRed1 (Matz et al., 1999
) make it an obvious choice for the generation of an MV persistently infected cell line for superinfection experiments. Furthermore, red fluorescent proteins are the proteins of choice for use in two colour fluorescence microscopy with EGFP, which is expressed by the recombinant virus MVeGFP (Duprex et al., 1999b
). A second recombinant MV (MVDsRed1) was therefore generated and, as expected, the level of fluorescence in syncytia was significantly less than that observed for MVeGFP, making it an ideal tool for the long-term labelling of persistently infected cells. Most importantly, these two viruses can be readily distinguished by fluorescence microscopy in living cells over time and are antigenically indistinguishable.
In this study, we used MVs expressing DsRed1 and EGFP to characterize true persistent infections. We demonstrated that persistence can be maintained in the presence of fully functional viral fusion complexes and that cell surface reorganization of the MV receptor CD46 plays an important role in this process. This is also the first investigation that uses this approach to examine viral persistence and superinfection immunity and it could readily be extended to other viruses that establish such infections. Superinfection immunity is extremely effective in preventing gene expression of related viruses.
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METHODS |
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Generation of MVDsRed1.
The open reading frame of DsRed1 was amplified from pDsRed N1 (BD Biosciences) using oligonucleotides priRFPMluI (5'-ATCCCGACGCGTACGCCACCATGGTGCGCTCCTCCAAGAAC-3') and priRFPNruI (5'-AAGCGATCGCGAGTCAGTCTACAGGAACAGGTGGTGG-3'). These primers contain MluI and NruI restriction sites (underlined), respectively, which permit the insertion of the resulting 695 bp fragment into similarly restricted pMeGFPNV, a plasmid which contains the gene encoding EGFP flanked by MluI and NruI (Hangartner, 1997). The resulting full-length anti-genomic clone of MV (pMDsRed1NV) contains an additional transcription unit of 810 nt and the genomic sequences conform to the rule of six (Calain & Roux, 1993
). A recombinant virus (MVDsRed1) was recovered from this plasmid by using the 293-3-46 cell line, which stably expresses T7 polymerase and the N and P proteins of MV. Cells were monitored daily for cell-to-cell fusion, and UV microscopy was used to determine if the resulting syncytia were autofluorescent. MVDsRed1 stocks were produced following plaque purification and titres of up to 106 TCID50 ml1 were obtained. Virus stocks were stored at 70 °C.
Growth analysis.
Vero cells were cultured to a confluency of 90 % in 25 cm2 tissue culture flasks. Cells were infected at an m.o.i. of 0·1 with MVDsRed1 for 1 h at 37 °C. The inoculum was removed and the cells were washed six times using PBS. Infected cells were incubated for 64 h and every 8 h the supernatant was removed and the cell sheet was scraped from the infected flasks into serum-free DMEM (5 ml). Cell-associated virus was recovered by freezethawing the cell samples twice. Titres were obtained using the 50 % end-point dilution assay.
Generation of persistently infected cell lines.
NT2 cells were infected at an m.o.i. of 0·01 with either MVeGFP or MVDsRed1. This resulted in the destruction of the majority of the cell monolayer by 5 days post-infection (p.i.). Growth medium was added every 3 days and the single autofluorescent cells that remained were observed to divide by 2 weeks p.i. At 6 weeks p.i., the resulting single colonies of autofluorescent cells were trypsinized and replated into 25 cm2 flasks. Confluent cell monolayers were obtained at 8 weeks p.i. The persistently infected cell lines were routinely split (1 : 3) two times per week. Cell lines were monitored regularly for autofluorescence by UV microscopy to ensure that true persistence had been attained and was continually maintained.
Indirect immunofluorescence and confocal microscopy.
Cell lines were grown on glass coverslips to a confluency of 80 % after which time they were rinsed twice in PBS. Cell surface expression of viral glycoproteins and the MV receptor CD46 was examined by incubating cells with the appropriate antiserum prior to fixation. The H glycoprotein was detected using monoclonal antibody (mAb) L77 (diluted 1 : 100 in PBS) (Moeller et al., 2001) and the F glycoprotein was detected using a polyclonal antiserum (diluted 1 : 500 in PBS), which were gifts from Jürgen Schneider-Schaulies, University of Würzburg, Germany. CD46 was detected using a polyclonal antiserum (diluted 1 : 100 in PBS), which was a gift from Fabian Wild, Institut Pasteur de Lyon, France. Cells were incubated for 1 h at 37 °C in the presence of the primary antibodies, after which time unbound antibodies were removed by three successive PBS washes. Cells were fixed for 10 min in 4 % (w/v) paraformaldehyde and the coverslips were rinsed in PBS. Viral antigens were detected intracellularly in paraformaldehyde fixed cells using mAb L77, a polyclonal antiserum to the cytoplasmic tail of the F glycoprotein (diluted 1 : 500 in PBS) and a mAb directed against the MV nucleocapsid-protein (diluted 1 : 1000 in PBS; Seralabs). Appropriate fluorescently labelled secondary antibodies were used, as previously outlined, to detect bound primary antibodies and a Leica TCS/NT confocal scanning laser microscope (CSLM), equipped with a krypton/argon laser as the source of the ion beam, was used to examine the samples (Duprex et al., 1999b
). DsRed1 was visualized by virtue of its autofluorescence by excitation at 568 nm with a 564596 nm band pass emission filter.
Live-cell confocal microscopy.
MVeGFP and MVDsRed1 acutely infected Vero and NT2 cells, persistently infected (pi) NT2-MVeGFP and piNT2-MVDsRed1, and superinfected cells were examined for fluorescence by CSLM as described previously (Duprex et al., 1999b).
Immunoblotting.
Vero, CHO, CHO-CD46, NT2 and piNT2-MVeGFP cells were lysed in Tris-glycine SDS sample buffer in the absence of a reducing agent. Western blots were performed by following standard procedures. CD46 was detected using an anti-CD46 rabbit polyclonal antibody (diluted 1 : 250 in PBS). An anti-rabbit immunoglobulin G (Fc) alkaline phosphatase conjugate (Promega) was used as a secondary antibody (diluted 1 : 5000 in PBS). Bound antibody was detected by immersing the gel in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (NBT/BCIP; Promega).
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RESULTS |
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MV glycoproteins traffic to the cell surface
The piNT2-MVeGFP cells were examined using antibodies to MV proteins to determine if there were any changes in the intracellular localization of the N protein, the major structural component of the ribonucleoprotein complex or the cell surface distribution of the H and F envelope glycoproteins. Intracytoplasmic inclusions, typical of acutely MV-infected cells, were observed in permeabilized cells stained for the N protein (Fig. 4a). The H and F glycoproteins were readily detected in the cytoplasm and at the cell surface of permeabilized cells using a monoclonal anti-H antibody and polyclonal anti-F cytoplasmic tail antiserum (Fig. 4a
). Unfixed, non-permeabilized piNT2 cells were examined by ICC to determine if the glycoproteins reached the cell surface. Both H and F were readily detected, indicating that there was no intracellular sequestration of the glycoproteins (Fig. 4b
). As these antibodies recognize conformational epitopes these data suggest the proteins are correctly folded, cleaved and processed within the cell. As N is exclusively a cytoplasmic protein, no reactivity was observed when an anti-N mAb was used on non-permeabilized cells (Fig. 4b
). In contrast to acutely infected NT2 cells, which release low levels of virus (
101·5 TCID50 ml1), released virus was not detected in supernatants from persistently infected cells.
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DISCUSSION |
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This investigation was undertaken to determine the degree of superinfection immunity in a disease relevant persistently infected human cell line using a fundamentally different approach to those used previously. Although superinfection immunity has been examined for many viruses, low levels of infection can never be ruled out as these studies have been restricted by the sensitivity of the detection methods employed (Bock et al., 1998; Breiner et al., 2001
; de la Torre et al., 1985
; Ho & Babiuk, 1979
; Menna et al., 1975
; Singh et al., 1997
; Walters et al., 2004
; Williams et al., 1981
). Without having to rely on observing an overt cytopathic effect we have proven conclusively that superinfection immunity is extremely strong and can be overcome in only 1 : 300 000 cells. Interestingly, these few cells continued to divide to generate small foci of co-infected cells, illustrating the dominant effect that initial virus used to generate the persistently infected cells has on the superinfecting virus. Therefore, fluorescent persistently infected cell lines have resolved this question conclusively for the prototype morbillivirus. Finally, MuV superinfection of the piNT2 cells proves that superinfection immunity is not mediated by a general, soluble antiviral factor.
From a wider perspective these data allow us to propose particular mechanisms that make persistence possible and exclude those which are clearly not involved. For example, attachment interference due to desialylation, which has been described for Newcastle disease virus, Sendai virus and Human parainfluenza virus 3 (Horga et al., 2000; Morrison & McGinnes, 1989
), can be ruled out as neuraminidase activity has not been demonstrated for MV, whereas attachment interference, due to receptor relocalization, could restrict MV entry. As infectious virus is released from the piNT2-overlaid Vero cell monolayers persistence also does not depend on the generation of mutations in the virus genome that inactivate assembly of virions. Furthermore, the fact that the F and H glycoproteins on the piNT2 cell surface are fusion competent demonstrates that loss of their function is not an essential step in the establishment of persistent MV infections. This correlates well with what is observed in SSPE viruses where alterations in the cytoplasmic tail of the F protein enhance, rather than abolish, fusion (Cathomen et al., 1998
). As glycosylation and furin cleavage of F0 are necessary for H and F1/F2 to form an active fusion complex it is also clear that these two processes are not impaired in piNT2 cells. Collectively, these data demonstrate that cellular factors are more important in the control of cell-to-cell fusion than mutations in the virus genome.
Receptor downmodulation and superinfection immunity have been examined extensively for a number of retroviruses. This phenomenon, originally identified in studies using Rous sarcoma virus (Rubin, 1960), is referred to as superinfection interference. Studies using human immunodeficiency virus (HIV) type 1 have shown that shortly after infection the primary receptor (CD4) is downmodulated from the cell surface. This is mediated by the Nef protein, which enhances CD4 endocytosis and lysosomal degradation (Garcia & Miller, 1991
). CD4 is also bound by nascent gp160 synthesized within infected cells. This leads to an intracellular sequestration of the protein, ensures that the virus receptor is not incorporated into budding virions and thus enhances the infectivity of the progeny virions (Bour et al., 1991
). Thus, loss of CD4 from the cells makes them fully or partially resistant to infection by other retroviruses that utilize this receptor. Similar data have been obtained using amphotropic murine leukaemia virus (A-MuLV), which uses a sodium-dependent transporter (Pit-2) as a receptor. Upon A-MuLV infection, Pit-2 is downmodulated from the cell surface to intracellular aggregates and this leads to superinfection interference (Jobbagy et al., 2000
). It is clear from our data that the mechanism of superinfection immunity in the persistent infections described in this report is fundamentally different for MV as we see neither internalization nor degradation of CD46. Rapid downregulation of CD46 from the cell surface of MV-infected cells was observed in the seminal studies that identified CD46 as an MV receptor (Naniche et al., 1993a
). The mechanisms of CD46 removal from the cell surface by MV have not been examined in detail though some studies have suggested that CD46 is not endocytosed (Maisner et al., 1997
; Teuchert et al., 1999
). Recently, two pathways that lead to CD46 internalization have been characterized (Crimeen-Irwin et al., 2003
). In the ligand-independent pathway, CD46 is constitutively internalized in clathrin-coated pits, and localizes to multivesicular perinuclear inclusions from where it is transported back to the plasma membrane. In the second ligand-dependent pathway, binding of either an anti-CD46 multivalent antibody or MV leads to a process similar to macropinocytosis, which in turn causes the degradation of plasma membrane bound CD46. CD46 downregulation is minimal in piNT2 cells and there is no detectable decrease in overall expression levels when the protein is examined biochemically and microscopically. In contrast to a previous study, which examined CD46 downregulation in persistently infected monkey fibroblasts using only biochemical assays (Hirano et al., 1996
), we consider that this study highlights the value of combining a cell biological approach, to examine CD46 localization, alongside a biochemical approach, to examine total levels of CD46. In piNT2 cells the F/H complexes present on the surface effect fusion only with cells that express MV receptors. A possible explanation is that CD46 is sequestered into specific domains at the plasma membrane, such as lipid rafts or caveolae, and that this renders the molecule incapable of acting as a receptor for the F/H complexes on neighbouring piNT2 cells. Whether CD46 relocalization plays a role in superinfection immunity remains to be determined. However, the fact that CDV could not also superinfect the piNT2 cells argues against this, as this virus does not use primate CD46, although whether canine CD46 can be utilized remains an open question. At present, we are attempting to generate a vaccine-based recombinant CDV, which expresses EGFP from an additional transcription unit to examine CDV superinfection using an equivalent approach. Mutations in domain 1 of the poliovirus receptor have been demonstrated to increase the resistance of persistently infected human neuroblastoma cells to virus mediated lysis (Pavio et al., 2000
). Hence, we are determining the sequence of CD46 in the piNT2 cells to examine this potential determinant.
The utility of recombinant viruses that express fluorescent proteins to study persistence has been clearly demonstrated in this study. Persistence does not depend on the generation of mutations in the virus genome, inactivation of the fusion complex on the cell surface, perturbation of M protein/ribonucleoprotein interactions or a general diminution in viral gene expression. This approach could be applied to any RNA or DNA virus for which there are rescue systems available, for example, it could be used to study HIV infections, where questions of superinfection interference and receptor downregulation are highly relevant (Altfeld et al., 2002; Locher et al., 1997
) and where persistently infected cells and EGFP-expressing recombinant viruses have been generated (Mahlknecht et al., 2000
; Tanaka et al., 1999
). Replication-defective Sindbis viruses (SINV), which express either EGFP or DsRed1 with a palmitoylation signal at the amino terminus of the fluorescent protein, have been produced. The palEGFP SINV was useful in vivo as an anterograde and retrograde neuronal tracer and it produced Golgi apparatus-like labelling of neurones, whereas the DsRed1 equivalent was unsuitable for this purpose due to a significant amount of fluorescent protein aggregation (Furuta et al., 2001
). Furthermore, SINV has been shown to persist in the brains of mice for up to 17 months (Levine & Griffin, 1993
). It would be interesting to ascertain if the DsRed1-expressing SINV could be used in an analogous manner to MVDsRed1 not only to generate persistently infected cell lines but to facilitate studies into the long-term persistence of the virus in vivo using this animal model. Finally, fluorescent laser capture microdissection is a new method that can be used to select and procure cell clusters from tissue sections and cell monolayers (Suarez-Quian et al., 1999
). This methodology could allow the study of host genes that permit superinfection of single cells by targeted amplification of candidate host genes by RT-PCR. Such information could be useful in the development of general strategies for controlling virus infections.
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ACKNOWLEDGEMENTS |
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Received 22 March 2005;
accepted 25 April 2005.
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