Scientific Report 2005

Immunology

CCR5 and CXCR4, Receptors for HIV Type 1

D.E. Mosier, C. Pastore, A. Ramos, R. Nedellec, O. Hartley,* R. Offord,* M. Lederman**

* Centre Médical Universitaire, Geneva, Switzerland
** Case Western Reserve University, Cleveland, Ohio

HIV type 1 (HIV-1) is the cause of the AIDS pandemic. The first step in HIV-1 infection is sequential binding of the virus to the cell-surface receptors CD4 and CCR5. Because CCR5 binding occurs after CD4 binding, CCR5 is defined as a coreceptor. The importance of CCR5 in HIV-1 infection was first appreciated because some persons have a natural mutation that prevents expression of CCR5. These persons are naturally resistant to HIV-1 infection, and they have no apparent clinical consequences of lacking CCR5. These observations led to research programs to develop CCR5-blocking agents to prevent HIV-1 infection.

HIV-1 can undergo mutations that allow a second chemokine receptor, CXCR4, to replace the binding function of CCR5. We have studied the costs to viral fitness of those mutations.

Antiviral Compounds that Target CCR5

The normal function of CCR5 is to bind chemokines and signal cell migration. RANTES is the CCR5-binding chemokine with the most potent activity against HIV-1, but it is poor at inhibiting the infection of macrophages. We prepared synthetic modifications of the N-terminal domain of RANTES. We found that the most potent of these compounds, PSC-RANTES, is 1000 times more effective than native RANTES at inhibiting HIV-1 infection and that it completely blocks infection of macrophages. A single injection of PSC-RANTES before inoculation of virus prevents HIV-1 infection of 100% of mice with severe combined immunodeficiency repopulated with human peripheral blood leukocytes. Brief exposure of human cells to PSC-RANTES leads to prolonged internalization of CCR5.These properties led to the formulation of PSC-RANTES as a topical microbicide to prevent sexual transmission of HIV-1. Recently, treatment with PSC-RANTES prevented vaginal transmission of a chimeric virus consisting of simian immunodeficiency virus and HIV in the rhesus macaque model. Preclinical development of this compound is progressing toward the first trials in humans.

Mutational Costs of Coreceptor Switching

One concern about CCR5-blocking agents such as PSC-RANTES is that they might select for resistant viruses that can infect via other chemokine receptors, such as CXCR4. Although previously we showed that such “coreceptor switch” mutants can arise during treatment, a recent detailed analysis revealed that most mutants have a loss of fitness during coreceptor switching that coincides with a period when neither CCR5 nor CXCR4 supports efficient virus infection.

The mutations in the HIV-1 envelope that drive coreceptor switching occur mainly in the exposed variable loops (V1/V2 and V3), and different HIV-1 isolates require as few as 1 mutation or as many as 7 mutations to switch from use of CCR5 to use of CXCR4. Poor replication in both CCR5- and CXCR4-expressing target cells and increased sensitivity to both CCR5 and CXCR4 inhibitors were common features of viruses that were switching coreceptors.

To more fully understand the cost of each mutation associated with changing coreceptor binding from CCR5 to CXCR4, we reconstructed all possible mutational pathways between a parental CCR5-using virus and a CXCR4-using descendent virus separated from the parent virus by 5 mutations. We used site-directed mutagenesis to introduce all 32 possible combinations of single and multiple mutations in the HIV-1 envelope gene. These mutated envelopes were combined with an envelope-deficient reporter virus to make HIV-1 particles capable of only a single cycle of infection.

We found that mutations in variable loops 1 and 2 of the envelope improved the use of CCR5 but did not permit infection via CXCR4. Mutations in variable loop 3 led to use of CXCR4 for viral entry, but only poorly. Combinations of mutations in all 3 variable loops improved the ability of the virus to use CXCR4. The sequence in which mutations were introduced was critical. About 30% of possible mutations were noninfectious.

Maximum likelihood analysis indicated 1 favored mutational pathway in 120 sequential possibilities. The probability of coreceptor switching is thus constrained by having to make the right mutation at the right place at the right time. In the lottery of ongoing viral mutation, a coreceptor switch event is a rare winner.

Publications

Hartley, O., Gaertner, H., Wilken, J., Thompson, D., Fish, R., Ramos, A., Pastore, C., Dufour, B., Cerini, F., Melotti, A., Heveker, N., Picard, L., Alizon, M., Mosier, D., Kent, S., Offord, R. Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors. Proc. Natl. Acad. Sci. U. S. A. 101:16460, 2004.

Lederman, M.M., Veazey, R.S., Offord, R., Mosier, D.E., Dufour, J., Mefford, M., Piatak, M., Jr., Lifson, J.D., Salkowitz, J.R., Rodriguez, B., Blauvelt, A., Hartley, O. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5. Science 306:485, 2004.

Mosier, D.E. HIV-1 envelope evolution and vaccine efficacy. Curr. Drug Targets Infect. Disord. 5:171, 2005.