Susceptibility to virus–cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

Abstract

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of virus-cell fusion confirming the correlation between post-translational modification of tubulin and virus-cell fusion. These results thus identify tubulin and its post-translational modification as a new cellular target for interference with HIV-cell fusion.

The Histone Deacetylase Inhibitor Vorinostat (SAHA) Increases the Susceptibility of Uninfected CD4 + T Cells to HIV by Increasing the Kinetics and Efficiency of Postentry Viral Events

“…Previous studies have found that inhibition of HDAC6 enhances HIV infection by increasing fusion in cell-cell and virus-cell fusion models (41,42), results that we were unable to replicate here. Differences in the viral or cell models used may account for these differences.…”

“…Our observation that vorinostat increased the kinetics of reverse transcription in CD4 ϩ T cells suggested that inhibition of cytoplasmic HDACs might contribute to increased susceptibility to HIV. Of these, the class IIb HDAC6 was of particular interest, because specific inhibitors have previously been reported to enhance HIV infection; however, these studies reported enhancement of viral fusion (41,42), which we did not observe with vorinostat treatment.…”

HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

“…It is thought that certain enveloped viruses such as herpes simplex virus 1 (HSV-1), Sendai virus, human immunodeficiency virus type 1 (HIV-1), and many retroviruses have pH-independent viral fusion proteins that allow the virus to penetrate into cells by fusing directly with the plasma membrane (Stein et al , 1987; Earp et al , 2005; Kielian and Rey, 2006; Marsh and Helenius, 2006). HIV-1 interacts with target cells through cell-surface CD4 and CXCR4 or CCR5 coreceptors, a process that is cooperative and requires cell signaling such as actin polymerization, reorganization (Iyengar et al , 1998; Jimenez-Baranda et al , 2007; Yoder et al , 2008; Barrero-Villar et al , 2009; Liu et al , 2009), and stabilization of microtubules (Valenzuela-Fernandez et al , 2005; Malinowsky et al , 2008) to achieve pore fusion formation. Although cytoskeleton reorganization and dynamics have well-documented roles in HIV-1 fusion and entry events, the contribution of plasma membrane dynamics is less clear during these early viral infection steps.…”

Overexpression of Histone Deacetylase 6 Enhances Resistance to Porcine Reproductive and Respiratory Syndrome Virus in Pigs

“…Thus, it was proposed that HDCA6 is a potential regulator of viral life cycles. With respect to HIV, the overexpression of HDAC6 reduces viral infection and decreases virus–cell membrane fusion by reducing Actub levels [41,19]. In IAV-infected cells, the virus induces microtubule acetylation to facilitate its intracellular trafficking [37].…”