Viral entry into a host cell requires the presence of specific receptors on the host cell surface. As a result, a given virus can only infect cell types that express the right receptor(s). The devastating human immunodeficiency virus HIV-1 recognizes cells that display the glycoprotein CD4. CD4 binding triggers a conformational change in the HIV-1 envelope protein (Env) such that it can then bind a co-receptor, either CCR5 or CXCR4 depending on the stage of infection (Figure 1). These virus-receptor interactions lead to fusion of the viral and cell membranes, allowing the virus to access the host cytoplasm.
Image provided by John Nahabedian
Understanding what features of CD4, CCR5 and CXCR4 facilitate HIV-1 entry can inform development of strategies to prevent HIV-1 infection. Toward this goal, researchers often use non-human primates as models because their cell receptors are similar to human receptors yet vary in their ability to allow HIV-1 entry. For example, “humans and owl monkeys, but not macaques, encode a CD4 receptor that, when paired with human CCR5, permits entry of transmissible HIV-1 variants due to a single residue difference” says John Nahabedian, a graduate student in the Overbaugh Laboratory (Human Biology Division). “However, little is known about whether different CCR5 receptor proteins act as determinants of host range.” In work recently published in Virology, Mr. Nahabedian and his colleagues in the Overbaugh lab set out to determine whether owl monkey CCR5 can function as a co-receptor for HIV-1.
In their first experiment, the researchers engineered human 293-T cells to express different combinations of CD4 and CCR5 from humans, owl monkeys or macaques and then challenged these cells with fluorescent HIV-1 viruses encoding one of four different Env variants. Infection was measured by counting fluorescent (i.e., virus-containing) cells via flow cytometry. As expected, cells expressing human forms of both CD4 and CCR5 were highly infection-prone in all cases, while cells expressing macaque CD4 and human CCR5 were far less susceptible to HIV-1 infection. For cells expressing both CD4 and CCR5 from owl monkeys, three of the four HIV-1 variants were completely unable to enter the cells. However, with two of the four viruses, pairing either owl monkey CD4 with human CCR5 or human CD4 with owl monkey CCR5 allowed HIV to enter the cells with similar efficiency as cells expressing both human proteins. This result indicates that whether owl monkey CD4 and CCR5 can serve as functional receptors for HIV-1 depends on the sequence of the other receptor, which was unexpected because it is commonly thought that CD4 and CCR5 do not interact with each other.
Owl monkey and human CCR5 share 92% amino acid identity, yet differ in their ability to function as a co-receptor for HIV-1. As a result, the authors could hone in on which amino acids account for the difference in receptor function. Because positions 15 and 16 of CCR5 differ between humans and owl monkeys and were already thought to be important for co-receptor activity of human CCR5, the authors tested whether mutating these amino acids in the owl monkey protein to match the human protein increases its ability to allow HIV-1 entry. Indeed, mutation of both residues to the human form was sufficient to confer human-level receptor function to owl monkey CCR5, when paired with human CCR4. Based on this result, the authors could conclude that positions 15 and 16 of CCR5 are necessary for its co-receptor function, possibly because they are near sulfated tyrosines at positions 10 and 14 that interact directly with HIV-1 Env.
In each of their experiments, the researchers noted that the behavior of owl monkey CCR5 depended on which HIV-1 variant the cells were challenged with. Thus, the ability of CCR5 to function as a co-receptor for HIV-1 depends not only on the characteristics of the CD4 protein present, but also on the characteristics of the HIV-1 Env protein. Although there was no apparent pattern to suggest which features of Env are important in determining its ability to bind owl monkey CCR5, the authors suggest that Env proteins with a more “open” conformation might be able to bind a wider range of CCR5 proteins.
The results of this study highlight just how nuanced the determinants of viral entry are. Fortunately, the efforts of the Overbaugh lab have brought the picture into better focus for CCR5. Their findings reveal that two specific amino acids in CCR5 are essential to its functionality as a co-receptor for HIV-1, and that owl monkey CCR5 can be used by HIV-1 in certain contexts. Future experiments will seek to expand these findings; according to John Nahabedian, “given that there is considerable within-species polymorphism in many genes relevant to viral replication, it is possible that there are additional alleles of owl monkey CCR5 that demonstrate improved function as a HIV-1 receptor.” Hopefully, studying these variants will continue to enhance our understanding of how a deadly virus gains access to host cells.
Nahabedian J, Sharma A, Kaczmarek ME, Wilkerson GK, Sawyer SL and Overbaugh J. “Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entry.” Virology. 2017 Dec;512:180-186. doi: 10.1016/j.virol.2017.09.018.
This research was supported by the National Institutes of Health, the Burroughs Wellcome Fund and the National Science Foundation.