Murine coronavirus with an extended host range uses heparan sulfate as an entry receptor - PubMed (original) (raw)
Murine coronavirus with an extended host range uses heparan sulfate as an entry receptor
Cornelis A M de Haan et al. J Virol. 2005 Nov.
Abstract
Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif. The adaptation of the virus through the use of heparan sulfate as an attachment/entry receptor was demonstrated by increased heparin binding as well as by inhibition of infection through treatment of cells and the virus with heparinase and heparin, respectively.
Figures
FIG. 1.
Genomic organization of the recombinant viruses. (A) The genome structures of the recombinant MHVs containing either the parental MHV-A59 or the MHV/BHK spike gene and an FL expression cassette are depicted (MHV-2aFLS and MHV-2aFLSrec, respectively). Numbers and lowercase letters designate the genes encoding nonstructural proteins, while genes encoding spike (S) protein, envelope (E) protein, membrane (M) protein, or nucleocapsid (N) protein are marked by the protein abbreviation. The 5′ and 3′ untranslated regions (UTR) are also indicated. (B) The spike protein is depicted as an elongated box. Each vertical line in this box indicates an amino acid substitution in the MHV/BHK S protein compared to the parental MHV-A59 spike protein. The triangle indicates a 7-amino-acid insertion. The MHV-A59 S protein can be cleaved at the position of the arrow into an amino-terminal S1 and a carboxy-terminal S2 domain. Horizontal lines designate the approximate locations of the receptor-binding domain (RBD), putative fusion peptide (FP) (5), heptad repeat region 1 (HR1) and HR2, and the transmembrane domain (TM). The encircled numbers specify the heparin-binding consensus sequences, the locations of which are indicated by gray boxes, while their sequences are given below for the MHV-A59 and the MHV/BHK spike proteins. The amino acid insertions and substitutions in the MHV/BHK spike protein compared to the MHV-A59 spike protein are underlined. The heparin consensus sequences themselves are also shown (X, any amino acid; B, basic amino acid).
FIG. 2.
Infection of murine cells. (A) LR7 cells were inoculated with MHV-2aFLS or MHV-2aFLSrec at a multiplicity of infection of 1. At the indicated time points postinfection (pi), the cells were lysed and intracellular luciferase expression was determined by using a luminometer (values are expressed in relative light units, RLU). Standard deviations are indicated. (B) Plaque phenotypes of recombinant viruses on LR7 cells. At 18 h postinfection, cells were fixed with a 3% formaldehyde solution, after which the agar overlay was removed. After being permeabilized with 1% Triton X-100 in phosphate-buffered saline, viral antigen was detected with the anti-MHV serum k134 at a 1:400 dilution. Peroxidase-conjugated swine immunoglobulins to rabbit immunoglobulins (Dakopatts) were used as secondary antibodies at a 1:100 dilution. Peroxidase was visualized, using an AEC substrate kit from Vector laboratories. Pictures were taken using bright-field microscopy and a Nikon DS-L1 digital camera. (C) LR7 cells were infected with the recombinant viruses indicated on the top of the gel at a multiplicity of infection of 10. Cells were labeled for 3 h with 35S-labeled amino acids (Amersham), starting 5 h postinfection. At the end of the labeling period, culture media were collected and prepared for immunoprecipitation with the anti-MHV serum k134, followed by polyacrylamide gel electrophoresis, followed by fluorography, as previously described (10). The positions of the different viral proteins are indicated on the left, while the molecular mass marker is indicated on the right. Asterisks indicate higher-order structures of the M and/or N proteins.
FIG. 3.
Infection of human cells. (A) HeLa cells were inoculated with MHV-2aFLS and MHV-2aFLSrec, and intracellular luciferase expression was determined as described in the legend to Fig. 2A. (B) HeLa cells were infected with the recombinant viruses as described above. At 18 h postinfection, the cells were fixed and viral antigen was detected as described in the legend to Fig. 2B.
FIG. 4.
Interaction with heparan sulfate/heparin. (A and B) LR7 and HeLa cells were inoculated with the recombinant viruses as described in the legend to Fig. 2A, except that the cells had been pretreated with heparinase I for 1.5 h before the inoculation (A) or the recombinant viruses had been incubated with different concentrations of heparin for 1 h at 4°C (B). At 5 h post infection, the FL activity in the cultures was determined. Standard deviations are indicated. (C) The percentage of MHV virions adsorbed to heparin-agarose beads was determined by a Taqman reverse transcriptase PCR specific for viral genomic RNA, as previously described (11). The black bars (+ heparin) represent the results when virions were incubated with heparin prior to incubation with the heparin-agarose beads, and the white bars indicate the results when the virions were not incubated with heparin prior to incubation (− heparin). Standard deviations are indicated.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources