Can mCherry or ZsGreen1 fusion proteins affect Nullbasic's antiviral activity in CD4+ cells?

P Rozario, H Jin and D Harrich

QIMR Berghofer Medical Research Institute

Human immunodeficiency virus-1 (HIV-1), the causative agent of autoimmune deficiency syndrome (AIDS), is a major health problem world-wide resulting in approximately 5000 new infection cases daily. Because of the large number of fatalities consequent of HIV, the importance of treatment research is vital. As of now, HIV-1 infections are being effectively controlled by antiretroviral therapy (ART) which inhibits virus replication in infected individuals to undetectable levels. However, suspension of ART leads to a rebound in viral load. Nullbasic, a mutant of HIV-1 transactivator of transcription(Tat) protein, exhibits strong antiviral activity in lymphocytes through the inhibition of multiple steps of HIV-1 replication cycle. Recently, it was noted that the antiviral activity of Nullbasic in CD4+ primary cells varied following its fusion with fluorescent proteins mCherry and ZsGreen1. Nullbasic-mCherry resulted in a reduction of antiviral activity, a contrast seen with Nullbasic-ZsGreen1. In this study, we investigated if this was a result of either the conformational changes in Nullbasic imparted by the respective protein partners or the multimeric states of each fluorescent protein. To achieve this, a self-cleaving peptide T2A was used to allow Nullbasic to exist independently compared Nullbasic-mCherry/ZsGreen1. Additionally, dimeric and tetrameric forms of Nullbasic were obtained by the addition of synthesized GCN4 leucine zippers to Nullbasic and the antiviral activity was compared to its original monomeric form of Nullbasic. All of the constructed Nullbasic expression vectors were transduced into Jurkat cells and human CD4+ primary cells. Positive cells were selected by a cell sorter and infected with HIV-1. HIV-1 levels were measured by p24ELISA.