Viral Chemokine Receptors

When my lab sequenced the first chemokine receptors CXCR2 and CCR1 in 1991, the top BLAST hit for CCR1 was open reading frame (ORF) US28 of human cytomegalovirus (HCMV), indicating an obvious common ancestor and a possible example of gene piracy. Pox virologists had already identified virally encoded TNF and IFN-γ binding proteins, copied from the host and redeployed as cytokine scavengers and immune evasion factors; however, there were no precedents for G protein-coupled receptors in viruses or signaling viral immunoreceptor homologs. Tom Schall, then at Genentech, who had candidate CC chemokine receptor clones, learned about our CCR1 discovery and proposed a collaboration. I told him about the key features of CCR1: its specificity for CCL3 and CCL5, as well as monocytes and lymphocytes, and its sequence homology to US28 (1); however, he eventually wanted to pursue CCR1 independently, and published it in Cell in fall, 1992 along with data that US28 could bind to CCL2, CCL3, CCL4, and CCL5. A month earlier, our paper had been rejected by Cell for lack of sufficient binding data; however, we eventually published our paper in early 1993 in the Journal of Experimental Medicine (1), and the NIH received the patent for cloning CCR1. Meanwhile, the complete sequence of the non-human primate herpesvirus Herpesvirus saimiri appeared in 1992, revealing a CXCR2 homolog known as ECRF3. Sunil Ahuja, then a post-doc in my lab and now a Professor at the University of Texas at San Antonio, used our Xenopus oocyte expression system to show that ECRF3, like CXCR2, mobilized calcium in response to the ELR CXC chemokines CXCL1, CXCL7, and CXCL8, the first example of a virally encoded chemokine receptor that signaled (2). My long-time colleague JiLiang Gao, who was a post-doc in my lab at the time, then showed that US28 was also a calcium flux signaling receptor for the same chemokines that Schall’s group had found bound to US28 (3). Together these papers pioneered a new field of virally encoded chemokine receptors that has expanded as more herpesvirus and poxvirus genomes have been sequenced. In addition, many virally encoded chemokines and secreted chemokine binding proteins were later identified, along with information about structure, signaling pathways, biological functions, and potential disease connections. Together, this work demonstrated unequivocally that the chemokine system has been selectively and preferentially expropriated by these types of viruses; however, exactly why remains unresolved. At an evolutionary level, it is remarkable that the viral chemokine receptors could be so distantly related to mammalian receptors with which they share ligands, particularly since this was not the case for the human chemokine receptors known at the time, CXCR1, CXCR2, CCR1, and CCR2. We also began cloning mouse counterparts of the human receptors we were finding and noticed that the mouse–human orthologs were more distantly related than expected. I decided to investigate this systematically by doing an in silico study of the mouse–human orthologs then in the data base, and found that for the ~500 available sequence pairs, the distribution of divergence was highly heterogenous. Most orthologs had high homology, but the ones that did not were mostly immunoregulatory factors. I published a paper in Cell describing this exceptionalism and proposed that it might relate to evolutionary pressure imposed by the predilection of viruses for this type of host gene (4). We continued to use homology cross-hybridization to clone additional chemokine receptors, including, in 1994, one we first named CC CKR5 that was later renamed CCR5. Christophe Combadiere, a post-doc in my lab now with his own lab in Paris, actually cloned CCR3 and CCR5 cDNAs from the same screen. He determined their sequences and leukocyte specificities in parallel, then investigated their chemokine specificities sequentially, starting with the eosinophilselective CCR3. We reported that CCL3, CCL4, and CCL5 were agonists for CCR3 in May, 1995, accepted by JBC 1 day after submission with no revisions (5). However, colleagues in the field, and ultimately we were skeptical since CCL3 and CCL4 lacked eosinophil activity. As a check, Christophe sequenced transfected cDNA from the original “CCR3” cell lines and, to our chagrin, found the CCR5 sequence, not CCR3. Apparently, the plasmid tube labeled “CCR5” had been mistaken for “CCR3”. We wrote a correction published in December, 1995 in JBC indicating that CCL3, CCL4, and CCL5 were agonists for a new receptor named CC CKR5. We then submitted a new paper with the CC CKR5 sequence, its RNA distribution, and ligands to JBC, which after an ~6 month review was rejected in part because the reviewers regarded it as partly duplicative of the CCR3 paper. The same month that our correction appeared in JBC, Paolo Lusso and colleagues in Bob Gallo’s lab at the NCI of NIH reported in Science that CCL3, CCL4, and CCL5, the signature ligands for CCR5, were able to suppress replication of macrophage