Three years ago this month, scientists made headlines when they discovered that HIV-1 first infects human immune cells by attaching not only to the well known CD4 protein displayed on the cell surface, but also to a second protein receptor called CCR5. While the discovery was heralded as a major new lead in AIDS research, scientists also recognized that much follow up would be needed to learn how to exploit the two proteins and stymie the virus.
This week, an international team of scientists offers an important new clue on how to do this. Reporting in the Proceedings of the National Academy of Sciences*, the researchers found that CD4 and CCR5 physically interact in the cell membrane, pointing to a potential new target to direct HIV therapies or vaccines. Currently, most anti-HIV treatments attack the virus after it already has entered immune cells.
"Our work shows that to enter immune cells, HIV-1 needs these proteins to remain in direct physical contact with each other," said Dimiter Dimitrov, Ph.D., Sc.D., a scientist at the National Cancer Institute in Frederick, Md., and senior author on the paper. "If one can prevent their association it might be possible to leave HIV with no way to get inside cells."
Dimitrov noted that inhibition of the CCR5-CD4 interaction in theory should have few, if any, side effects in people. Previous studies show that people who produce defective CCR5 protein are usually naturally resistant to HIV-1 infection, and yet they show no ill effects from lacking the protein, suggesting it is not vital to human health.
This week's finding builds on the growing research interest in chemokine receptors, a family of proteins - including CCR5 that reside on the surface of immune cells. In June 1996, scientists discovered that the strains of HIV-1 responsible for the initial, mostly asymptomatic, infection target human immune cells displaying both CD4 and CCR5.
As a follow up to this initial finding, some studies have hinted that CD4 and CCR5 might physically interact somewhere on the cell surface, an observation that suggested a target in the cell where the life cycle of the virus can be disrupted.
To test the observation that there may be naturally occurring complexes of membrane-associated molecules, Dimitrov and colleagues embarked on the current study. In a series of difficult experiments with laboratory cell lines and primary human immune cells, the group charted the patterns of interaction between four proteins: CD4, CCR5, the HIV-1 protein gp120 that initially binds the virus to human cells, and CXCR4, which serves as a coreceptor for strains of HIV-1 that occur frequently in the late stages of disease.
Dimitrov said his group found the interaction of CD4 with CCR5 was surprisingly stronger than that with CXCR4, which in previous work had been suggested as physically complexing with CD4 in the presence of gp120. He said that in contrast to CXCR4, the association between CD4 and CCR5 was so tight to begin with that it was not significantly increased by the binding action of gp120.
The scientists then took the next step and partially characterized some of the regions of each protein where the physical interaction takes place. Interestingly, when antibodies designed to target the interaction were introduced into cultured cells, they partially inhibited HIV-1's subsequent ability to mediate membrane fusion, which is an important component of viral entry into cells.
"We are now in a position to exploit our ability to produce and isolate CD4-CCR5-gp120 complexes for use of this trimolecular complex to generate an AIDS vaccine," says Christopher Broder, Ph.D., a scientist at Uniformed Services University of the Health Sciences in Bethesda, Md., and an author on the study. "Experiments are under way to test the efficacy of such a vaccine in animals."
According to Xiaodong Xiao, Ph.D., an NCI scientist and the study's lead author, these results support a hypothesis proposed by Robin Weiss, a noted scientist at the Institute for Cancer Research in London. Weiss suggested that CCR5, originally identified as a secondary receptor, may have been the primary receptor for a viral ancestor of HIV-1. In fact, previous studies show that several strains of simian immunodeficiency virus (SIV), which share a common ancestor with HIV-1, can enter cells that lack CD4 by latching onto CCR5 alone.
"By showing such a strong intrinsic association between these two receptors, the notion of a possible evolutionary pathway in which variants of an older CCR5-dependent virus emerged that used both receptors to attach to immune cells is supported," said Xiao. "The use of CXCR4 as another coreceptor was probably a later evolutionary event, which interestingly correlates with the more frequent appearance of CXCR4-dependent HIV-1 strains in the late stages of the disease."
The scientists also said the results of this study may have even broader biological implications. "CCR5 is known in the field as a seven-transmembrane-domain, G-protein-coupled receptor," said Lijun Wu, Ph.D., a scientist at LeukoSite, Inc., in Cambridge, Mass. "CD4 does not belong to this protein family but to the immunoglobulin superfamily. Our finding is the first demonstration of a constitutive association between receptors from these unrelated families, suggesting a new and unexplored possibility for cross-talk between different types of cell surface receptors. It may provide another potential target for developing therapeutic agents against inflammatory responses."
This study involved a collaboration among scientists from NCI-Frederick, LeukoSite, Inc., Uniformed Services University of the Health Sciences, Centre d'Immunologie de Marseille-Luminy, and the Walter Reed Army Institute for Research.
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* The title of the study is "Constitutive Cell Surface Association Between CD4 and CCR5," article dated June 22, 1999. The authors are Xiaodong Xiao, Lijun Wu, Tzanko S. Stantchev, Yan-Ru Feng, Sophie Ugolini, Hong Chen, Zhimin Shen, James L. Riley, Christopher C. Broder, Quentin J. Sattentau, and Dimiter S. Dimitrov.