Background: CCR5 and CXCR4, are the primary coreceptors used by most HIV-1 strains. CCR3 has been implicated as a coreceptor for HIV infection in some cells, particularly the microglia cells of the central nervous system (CNS). To investigate its contribution to viral infection and to assess its potential as a target for gene therapeutic manipulation, we applied two approaches. 

Methods: A panel of interfering RNAs  targeting RNA (siRNAs) sequences shared by murine and human CCR3 transcripts was used to assess effectiveness of CCR3 downregulation and protection from CCR3-tropic strains of HIV. At the same time we sought to develop an SFv antibody to CCR3, derived from the hybridoma 5E8-G9-B4 was made using overlap PCR and cloned in an mammalian expression vector.

Results: Out of a panel of CCR3 siRNAs, R3-526, was found to be most effective in downregulating CCR3. Both R3-526 and SFv addition to cultures resulted in almost 80% decrease in CCR3 in U87.CD4.CCR3 cell line, primary human fetal brain microglia cells and in primary human monocyte-derived macrophages. Decreased CCR3 expression was greatest at siRNA concentrations of 1nM to 10nM. Both ScFv and  siRNA also significantly decreased (>70%) productive HIV p24 HIV infection in microglia cells when tested in ADA and a primary Brain isolate. Other strains of HIV-1 which are CCR5-tropic were not inhibited.

Conclusions: Since CCR3 and CCR5 are expressed in microglia cells, therapy targetting both may be useful in CNS AIDS. Thus, recombinant SV40 viruses of both the molecules are being tested for their ability to infect purified microglia cells and macrophages, and protect them from HIV infection. Utilization of CCR3 by these HIV-1 isolates and their susceptibility to inhibition by siRNA and ScFv in primary brain microglia/macrophages may both help clarify the role of CCR3 as a coreceptor in CNS establishing HIV-1 and provide new avenues for therapeutic manipulation.