The presence of unintegrated viral DNA has been reported in cells persistently infected by lentiviruses, including human immunodeficiency virus type 1 (HIV-1). We confirm that CD8 + cells can be productively and persistently infected by HIV-1 for up to 4 months, as determined by secretion of viral core antigen p24 into the extracellular medium and by indirect immunofluorescence. The expression of the external viral glycoprotein gpl20 at the surface of these cells was demonstrated by two-colour flow cytometry. Progeny virions recovered from CD8 ÷ cells were infectious in CD4 ÷ T cells. Despite an absence of significant cytopathology, these chronically infected CD8 ÷ cells were shown to harbour unintegrated HIV-1 DNA, as detected by quantitative PCR. Both linear and circular forms of the extrachromosomal viral genome were present in infected CD8 ÷ cells, as early as 3 weeks before a peak in viral replication. These findings provide evidence that the presence of unintegrated viral DNA during lentiviral infection may not always correlate with c.p.e.

Several groups have shown that peripheral CD8+ lymphocytes can be infected with human immunodeficiency virus type 1 (HIV-1), resulting in noncytopathic infection and persistent production of viral particles. We studied the ability of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddl) to inhibit the establishment of HIV-1 infection in CD8+ cells that were derived from cultures of peripheral blood lymphocytes exposed to both virus and drug. In situ infection of CD8+ cells was demonstrated by double flow cytometry analysis by using both anti-glycoprotein 120 (anti-gp120) and anti-CD8 monoclonal antibodies. At higher concentrations of drug (e.g., 0.4 ,uM AZT), the production of viral particles was inhibited for over 2 months, as assessed by p24 antigen levels in the culture medium. We also performed a time course experiment to determine whether HIV-1 infection of CD8+ cells would be affected by treatment of peripheral blood lymphocytes with AZT or ddl for different intervals following exposure to virus. Quantitative PCR revealed that 0.4 ,uM AZT, added as late as 24 h after infection, interfered with the formation of proviral DNA in CD8+ cells. Both HIV-1 load and the production of progeny virions by CD8+ cells, as monitored by reverse transcriptase activity in culture fluids, were inhibited by both AZT and ddl in a dose-dependent manner.

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