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Background Transcription of HIV-1 cDNA prior to, or in the absence of, integration leads to synthesis of all classes of viral RNA transcripts. Yet only a limited range of viral proteins, including Nef, are translated in this context. Nef expression from unintegrated HIV-1 DNA has been shown to reduce cell surface CD4 levels in T-cells. We wished to determine whether Nef expressed from unintegrated DNA was also able to downregulate the chemokine coreceptors CXCR4 and CCR5. Viral integration was blocked through use of an inactive integrase or by using the integrase inhibitor raltegravir. Infected cells bearing unintegrated DNA were assayed by flow cytometry in the GFP reporter cell line, Rev-CEM, for cell surface levels of CD4, CXCR4 and CCR5. Results In cells bearing only unintegrated HIV-1 DNA, we found that surface levels of CXCR4 were significantly reduced, while levels of CCR5 were also diminished, but not to the extent of CXCR4. We also confirmed the downregulation of CD4. Similar patterns of results were obtained with both integrase-deficient virus or with wild-type infections of cells treated with raltegravir. The Alu-HIV qPCR assay that we used for detection of proviral DNA did not detect any integrated viral DNA. Conclusions Our results demonstrate that Nef can be expressed from unintegrated DNA at functionally relevant levels and suggest a role for Nef in downregulation of CXCR4 and CCR5. These findings may help to explain how downregulation of CXCR4, CCR5 and CD4 might restrict superinfection and/or prevent signal transduction involving HIV-1 infected cells.