The results described in this paper demonstrate the feasibility of redirecting recombinant murine coronavirus MHV to a nonnative receptor on human cells. To achieve this, we introduced bidirectional adapter protein-encoding genes, composed of the spike-binding domain of mCEACAM1a fused to a His tag targeting peptide into the MHV genome. This yielded a recombinant MHV that could establish a receptor-specific infection of sFvHis receptor-expressing human cells, resulting in extensive cell-cell fusion and efficient killing of the target cells.
Many viruses have been explored in various ways for their potential use as oncolytic agents. Several strategies successfully applied genetic modification of viruses to alter their receptor binding specificity and direct them to tumor cells. In some cases, targeting molecules were directly incorporated into the viral surface proteins (7
). Similar strategies were not effective for MHV and fMHV, as viable spike-modified viruses could not be rescued (our unpublished data). This is most probably due to the complex nature and dual function of the S protein, changes in which may easily lead to impairment of the conformational rearrangements in the spike essential for its functioning during cell entry. Soluble receptor proteins have been used to redirect replication-deficient adenoviruses (6
) and, very recently, herpes simplex virus (31
) to cancer cells. Strikingly, replication-competent adenoviruses were impaired in their oncolytic potential when expressing a soluble receptor targeting molecule (20
). Here we have demonstrated that the species barrier of coronaviruses can be alleviated, as shown by the insertion of adapter proteins into the MHV viral genome.
In general, soluble receptors, i.e., the functional fragments from otherwise membrane-anchored cell surface antigens acting as receptors, bind to viruses and neutralize virus infectivity. These features have been explored in quite some detail for murine coronaviruses (16
). It has been shown that the N-terminal domain of the mCEACAM1a receptor is sufficient for MHV-A59 binding and for induction of a conformational change in the S protein. This N-terminal domain can block virus infectivity for susceptible cells. Its expression on nonsusceptible cells is only sufficient to render cells susceptible to MHV once a second domain of the mCEACAM1a is also introduced (12
) or when it interacts with another unidentified molecule on the plasma membrane (10
). Here we demonstrated that when the N-terminal domain of mCEACAM1a is linked to a suitable peptide ligand, the fusion protein can function as an adapter protein that targets MHV to a corresponding receptor on a nonsusceptible cell. The subsequent establishment of infection functionally demonstrates that the adapter protein is sufficient to bind to and induce the conformational changes in the spike essential for MHV infection (15
The efficiency of MHV targeting to a nonnative receptor was critically dependent on the presence of a hinge region in the adapter protein. Adapter proteins containing such a region established a more effective infection of sFvHis-expressing target cells than similar adapter proteins lacking this region. The hinge region might function as an extended linker between the spike-binding domain soR and the target cell receptor, thereby affecting the conformation of the adapter protein or the spacing between the virus membrane and the cell membrane. That the distance between these membranes may be important was also suggested by the results of Dveksler et al. (12
), who found that, in contrast to expression of only the N-terminal domain of mCEACAM1a, extension of this domain with a second immunoglobulin-like motif rendered cells susceptible to MHV infection. Alternatively, the formation of disulfide-linked adapter protein dimers might account for the difference in targeting efficiency. Indeed, reduction of the dimer into a monomeric adapter protein by using dithiothreitol led to a drastic decrease in infected target cells (data not shown). Further research is needed to conclusively determine the role of the hinge region in the adapter protein.
The presence of the hinge region also had a pronounced effect on the yields of adapter protein-expressing recombinant MHV viruses from murine LR7 cells. While MHVsoR-His replicated to titers similar to that of the control virus MHVd2aHE, a severe reduction in yield was observed for MHVsoR-h-His (Fig. ). It is unlikely that the gene of soR-h-His, larger by merely 51 nucleotides, caused this difference. Since the amounts of the adapter proteins produced by the MHV recombinant viruses were approximately similar (Fig. ), it is more likely that the effect is the result of interactions between the adapter proteins and the spike proteins within the infected cell, which might lead to an impairment of virus release. Accordingly, we observed that soR-h-His neutralized MHV infection with murine LR7 cells more efficiently than did soR-His (53
). Apparently, the disulfide-linked dimer of soR has a higher affinity for the spike protein than the monomeric form and it thereby acts as a stronger competitor for binding of MHV to cellular mCEACAM1a. The subsequent reduction in entry of MHVsoR-h-His would explain the lower yield of this virus from mCEACAM1a-expressing cells.
The efficiencies with which our self-targeted MHVs could infect human target cells differed between the two HissFv-expressing cell lines: 293-HissFv.rec cells were the most susceptible, giving rise to extensive cell-cell fusion and ultimately eradication of cell cultures, which was not observed with U118MG-HissFv.rec cells. The significantly higher sFvHis receptor expression with 293-HissFv.rec cells probably accounts for this difference. As a high level of mCEACAM1a expression is also essential for an effective native infection of MHV (9
), a similar receptor concentration-dependent infection might apply to nonnative receptors as well. We cannot, however, exclude that other inherent differences between 293-HissFv.rec and U118MG-HissFv.rec cells are responsible for the observed different outcomes.