Virus receptors and tropism

Interestingly, endothelial cell-tropism was restored much earlier than leuko-tropism [ 34 , 35 ]. The identity of the laboratory and endothelial cell-tropic variants of both AD and Towne strains suggested that even a minor variation of the viral genome e.

Furthermore, the Toledo strain, at that time considered to be a prototype of wild-type HCMV strains, was shown to lack both endothelial cell- and leuko-tropism just like laboratory-adapted HCMV strains , whereas the endothelial cell-tropic Toledo variant possessed a highly-divergent genome, suggesting that it arose from an unrelated strain [ 36 ]. The investigation of the genetic determinants of endothelial cell- and leuko-tropism started in with a joint project by Italian and German researchers.

By cloning an endothelial cell-tropic HCMV isolate VR as a bacterial artificial chromosome, they generated a stable genetic material that was used in mutagenesis screens [ 37 , 38 ]. During the course of a two-year study, it became evident that it was the UL locus ULL that was essential for both HCMV endothelial cell- and leuko-tropism [ 42 ]. Several loss-of-function and gain-of-function experiments corroborated this conclusion. The second loss-of-function proof consisted of the detection of spontaneous mutations of natural viral variants within ULL, which were detrimental to both endothelial cell- and leuko-tropism.

In particular, eight independent HCMV clinical strains that had extensively propagated in fibroblast exhibited a series of spontaneous mutations, each affecting the coding sequence of one gene within the ULL. On the other hand, gain-of-function was documented by two experimental findings. The first consisted of the phenotypic reversion of multiple HCMV natural variant strains to both endothelial cell-tropism and leuko-tropism following reversal of mutations within the ULL.

This was routinely achieved through the propagation of fibroblast-passaged mutated strains in endothelial cells, with the rescue of the original coding sequence. The second finding was the partial rescue of both properties by trans-complementation with each of the three individual genes of ULL. The reason for this apparently unexplained result came from the Northern blot analysis, showing that the three genes of the ULL are all part of a single transcriptional unit [ 42 ]. Conversely, deletion of UL preserves the transcription of the upstream genes UL and UL and can be rescued by trans-complementation with UL After , when the indispensable role of ULL genes for endothelial cell-tropism was reported, several research groups investigated their role in epithelial cell-tropism.

Furthermore, it was shown that HCMV entry into epithelial cells and endothelial cells depends on genes belonging to the ULUL cluster and occurs by endocytosis and low pH-dependent fusion, unlike the pH-independent fusion of the virus envelope with the plasma membrane that occurs in fibroblasts [ 47 ].

These results were further extended when it was demonstrated that the expression of the PC in ARPE epithelial cells, but not in fibroblasts, caused resistance to HCMV infection [ 50 ].

Taken together, these findings confirmed the important role of the PC in endothelial cell-tropism of HCMV and, importantly, anticipated the presence of a pentamer receptor in epithelial cells.

Since its first isolation in the s, HCMV has been routinely recovered from and propagated in fibroblast cells. However, the mechanism of entry into fibroblasts is still under discussion. Recent observations suggest that it may occur via a rapid micropinocytosis [ 54 ] or even by direct fusion with the outer plasma membrane through the fusogenic activity of gB [ 55 ]. The primary candidates for this differential neutralizing activity appeared to be antibodies directed against ULL proteins, as indirectly suggested by murine and rabbit polyclonal antibodies raised against the individual components of pULL [ 44 , 56 , 57 ].

These results were due to the presence of a mutation in the ULL of Towne, while gB was found to possess a much lower potency in eliciting nAbs compared to PC [ 59 , 60 , 61 ]. Direct confirmation of these findings was obtained in , when the combination of memory B-cell immortalization and nAb determination in different target cells allowed researchers to identify two panels of human nAbs induced by natural infection [ 59 ].

It was found that complex assembly is promoted by the formation of disulfide bonds between the cysteine of gL and either the cysteine of gO TC or the cysteine of pUL PC , thus providing a molecular explanation for the modulation of cell tropism by these two glycoprotein complexes [ 62 ]. Using the same purified soluble forms of TC and PC employed in defining their molecular architecture, the binding sites for the potently neutralizing human mAbs mentioned above [ 59 ] were defined on their surfaces [ 64 ].

By means of mass spectrometry paired with chemical crosslinking, three neutralizing epitopes were identified in gH conserved in both TC and PC , along with five different neutralizing sites that were determined in the ULL portion of PC [ 64 ]. By , a series of discoveries had resulted in a thorough characterization of the PC and TC architecture.

Firstly, both gH-specific antibodies and the recombinant soluble TC inhibited viral infection of fibroblasts by AD Similarly, the mechanism of viral entry into epithelial cells was inhibited by anti-PC antibodies, as well as by the soluble PC. In addition, it was found that the anti-gO antibody was able to reduce ARPE cell infection, thus suggesting that the TC may exert a dual function of viral ligand and activator of gB fusogenic activity [ 61 , 69 ].

SDS-PAGE confirmed the composition of different complexes, which were then analyzed by transmission EM and negative staining with two-dimensional averaging. The shift in the elution time of b and c complexes compared to a is indicated by vertical dotted lines.

A previous study observed the activation of the ErbB family member by the PC, but did not detect direct binding between the PC and ErbB receptors [ 68 ]. Thus, ErbB activation could be a consequence of the Nrp2 pathway activation upon direct engagement with the PC [ 72 ]. The subsequent methodological steps leading to the final identification of cellular receptors for both the TC and PC are as follows.

Then, the PC was assayed using the new test system, which revealed that Nrp2 was the main interaction partner for PC in the single transmembrane panel. Other single transmembrane receptors were found to have different degrees of affinity with either the TC i. The role of these putative co-receptors needs to be further investigated. The structure of the Pentamer-Nrp2 complex, bound to the Fab fragment of the neutralizing mAb 3G16 [ 59 ], was studied using negative staining EM and single particle analysis, which revealed the 3D model of interaction between the viral gp complex and its cellular receptor Figure 3 a [ 72 ].

Additional cross-links were observed between the a2 and b1 domains and residues of pUL and pUL In addition, using chemical cross-linking coupled to MS analysis on the same samples that were used for EM analysis, the interaction of Nrp2 and PC was investigated Figure 3 b,c [ 72 ].

This analysis detected several intra- and inter-molecular interactions within the Pentamer-Nrp2 complex. In particular, it showed that the four domains a1-a2-b1-b2 of the extra-cellular region of Nrp2 interacted with distinct regions of the PC. These data suggested that the a1 domain rearranges itself in relation to the remainder of Nrp2 upon PC binding. Moreover, the authors confirmed by SEC that the combination of all four extracellular domains of Nrp2 was required for PC binding, since neither Nrp2-a1a2 nor Nrp2-a1b1b2 alone were able to bind it.

Mutations in pUL or pUL dramatically reduced the interaction with Nrp2, whereas mutations of pUL had no effect on binding [ 72 ]. Overall, this experimental evidence provided an in-depth insight into the features of the architecture of the PC-Nrp2 complex.

The setup of the experimental study was based on the presumption that, by knocking out genes that are important for HCMV entry, cells will become resistant to infection and, hence, survive.

To this end, epithelial cells were repeatedly exposed to HCMV for three months, and cellular clones resistant to viral infection underwent targeted sequencing, which identified OR14I1 and to a lesser extent Nrp2 as the gene required for productive HCMV entry [ 75 ]. We also find it intriguing that HCMV would use two pathways one pH-dependent and the other pH-independent to infect different cell types.

Thus, PC-Nrp2 interaction may be an alternative pathway for fibroblast infection. The identification of HCMV glycoprotein complexes and the cellular receptors involved in virus entry is fundamental in understanding the mechanisms of HCMV infection and neutralization.

This has prompted the development of novel strategies for the treatment and prevention of HCMV infection. In particular, the identification of the PC as a major determinant of HCMV cellular tropism and the most important target of human neutralizing antibodies supported the therapeutic use of the anti-PC antibody and the inclusion of the PC in vaccine formulations.

Recently, a phase II clinical trial showed that the administration of a combination of two mAbs one human to gH and one murine humanized to PC reduced HCMV infection and disease in high-risk kidney transplant recipients [ 80 ].

Currently, the most advanced vaccine approach to exploit the new knowledge on PC function is a live attenuated AD whole virion vaccine V , in which PC expression was restored by repairing a frameshift mutation in the UL gene [ 81 ].

Other promising vaccine formulations are at the preclinical evaluation stage. One example is a subunit recombinant PC vaccine that was produced in a secreted form from the hamster ovary cell line, which was stably transfected with a single polycistronic vector encoding the five PC genes; in mice, this was found to induce neutralizing antibody titers that were —fold higher than those induced by natural infection in humans [ 82 ]. More recently, a modified vaccinia virus Ankara-vectored vaccine composed of pp65, gB, and PC was evaluated in mice.

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Abstract The virus particles described in previous chapters are vehicles that transmit the viral genome and the infection from cell to cell. Publication types Research Support, N.