Abstract
BackgroundMacrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication.ResultsFollowing termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection.ConclusionWhile no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals.
Highlights
Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments
While we found no evidence for differential mRNA stability of tat mRNA in macrophages at any time after infection, changes in the balance between enhancing and inhibitory cellular splicing factors induced by infection suggest that regulation of HIV-1 alternative splicing plays a key role in persistent infection in these important viral reservoirs
HIV-1 replication in macrophages appears to be regulated at the level of tat mRNA expression
Summary
Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. Macrophages are one of the major target cells for HIV-1 in the body, are infected very early and remain an important reservoir of long-lived cells [1]. The proportion of tissue macrophages harbouring HIV-1 may be as high as 50% [6] and they become a major source of virus during opportunistic infection [1] or when CD4+ T cells are depleted [7]. In the gut-associated lymphoid tissue, the largest lymphoid organ in the body and the primary site for acute HIV-1 replication [8], macrophages are the predominant viral reservoir following massive depletion of CD4+ memory T cells during this acute infection stage [9]. Infection impairs vital macrophage functions such as phagocytosis, intracellular killing, cytokine production and chemotaxis [10]
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