Predicted membrane topology of the coronavirus protein E1

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.     

Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.     

Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formation of stress granules and processing bodies

Many viruses, including coronaviruses, induce host translational shutoff, while maintaining synthesis of their own gene products. In this study we performed genome-wide microarray analyses of the expression patterns of mouse hepatitis coronavirus (MHV)-infected cells. At the time of MHV-induced host translational shutoff, downregulation of numerous mRNAs, many of which encode protein translation-related factors, was observed. This downregulation, which is reminiscent of a cellular stress response, was dependent on viral replication and caused by mRNA decay. Concomitantly, phosphorylation of the eukaryotic translation initiation factor 2alpha was increased in MHV-infected cells. In addition, stress granules and processing bodies appeared, which are sites for mRNA stalling and degradation respectively. We propose that MHV replication induces host translational shutoff by triggering an integrated stress response. However, MHV replication per se does not appear to benefit from the inhibition of host protein synthesis, at least in vitro, since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff.     

A Single Point Mutation Creating a Furin Cleavage Site in the Spike Protein Renders Porcine Epidemic Diarrhea Coronavirus Trypsin Independent for Cell Entry and Fusion

The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein, we created a recombinant PEDV with an artificial furin protease cleavage site N terminal of the putative fusion peptide (PEDV-S^FCS). PEDV-S^FCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independently of trypsin, though to low titer.