Abstract
BackgroundThe regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized.MethodsTo better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes.ResultsIn activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues.ConclusionsCycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.
Highlights
The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages
Significant upregulation of cyclin T1 in activated human memory CD4 T cells To first characterize CycT1 protein expression of normal uninfected memory CD4 T cells by flow cytometry, memory CD4+CD45RO+ T cells were purified from peripheral blood of healthy donors and activated by CD3+CD28 mabs and IL2 for up to 5 days
The present study suggests that the return of infected activated CD4 T cells to quiescence is preceded by downregulation of CycT1 expression and Positive Transcription Elongation Factor b (P-TEFb) function, significantly restricting HIV replication and promoting latency establishment
Summary
The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. HIV-1 replication is notable for its strict dependence on host factors, of which Cyclin T1 (CycT1) is one of the most essential for viral RNA transcription in CD4 T cells and macrophages. CycT1 is a regulatory subunit that associates with the cyclin-dependent kinase CDK9 to form the Positive Transcription Elongation Factor b (P-TEFb), an enzymatic complex that phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAP II) to initiate mRNA transcriptional elongation [1,2,3]. The molecular details and role of P-TEFb and CycT1 for HIV transcription are well defined, but the interaction between CycT1 and HIV replication at single cell levels are less characterized
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