Related Experiment Video
Updated: May 31, 2026
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
Published on: March 8, 2012
The role of ITCH protein in human T-cell leukemia virus type 1 release
Batsukh Dorjbal1, David Derse, Patricia Lloyd
1HIV-Drug Resistance Program, NCI Frederick, Frederick, Maryland 21702, USA.
Abstract:
Human T-cell leukemia virus type 1 (HTLV-1) has two late domain (LD) motifs, PPPY and PTAP, which are important for viral budding. Mutations in the PPPY motif are more deleterious for viral release than changes in the PTAP motif. Several reports have shown that the interaction of PPPY with the WW domains of a Nedd4 (neuronal precursor cell-expressed developmentally down-regulated-4) family ubiquitin ligase (UL) is a critical event in virus release. We tested nine members of the Nedd4 family ULs and found that ITCH is the main contributor to HTLV-1 budding. ITCH overexpression strongly inhibited release and infectivity of wild-type (wt) HTLV-1, but rescued the release of infectious virions with certain mutations in the PPPY motif. Electron microscopy showed either fewer or misshapen virus particles when wt HTLV-1 was produced in the presence of overexpressed ITCH, whereas mutants with changes in the PPPY motif yielded normal looking particles at wt level. The other ULs had significantly weaker or no effects on HTLV-1 release and infectivity except for SMURF-1, which caused enhanced release of wt and all PPPY(-) mutant particles. These particles were poorly infectious and showed abnormal morphology by electron microscopy. Budding and infectivity defects due to overexpression of ITCH and SMURF-1 were correlated with higher than normal ubiquitination of Gag. Only silencing of ITCH, but not of WWP1, WWP2, and Nedd4, resulted in a reduction of HTLV-1 budding from 293T cells. The binding efficiencies between the HTLV-1 LD and WW domains of different ULs as measured by mammalian two-hybrid interaction did not correlate with the strength of their effect on HTLV-1 budding.
Insights
Human T-cell leukemia virus type 1 (HTLV-1) budding relies on late domain (LD) motifs interacting with Nedd4 family ubiquitin ligases. ITCH was identified as a key factor, inhibiting HTLV-1 release, while SMURF-1 enhanced it.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Human T-cell leukemia virus type 1 (HTLV-1) utilizes late domain (LD) motifs, specifically PPPY and PTAP, for efficient viral budding and release.
- The PPPY motif's interaction with WW domains of Nedd4 family ubiquitin ligases is crucial for HTLV-1 release, with PPPY mutations being more detrimental than PTAP mutations.
Purpose of the Study:
- To investigate the roles of different Nedd4 family ubiquitin ligases in HTLV-1 budding and release.
- To identify specific ubiquitin ligases that modulate HTLV-1 particle production and infectivity.
Main Methods:
- Screening of nine Nedd4 family ubiquitin ligases for their effects on HTLV-1 budding and infectivity.
- Overexpression and silencing experiments of selected ubiquitin ligases (ITCH, SMURF-1) in HTLV-1-producing cells.
- Electron microscopy to assess viral particle morphology and quantity.
- Analysis of Gag protein ubiquitination levels.
- Mammalian two-hybrid assays to measure binding affinities between HTLV-1 LD motifs and WW domains.
Main Results:
- ITCH was identified as the primary Nedd4 family ubiquitin ligase affecting HTLV-1 budding, with its overexpression inhibiting wild-type (wt) HTLV-1 release and infectivity.
- ITCH overexpression rescued the release of infectious virions from HTLV-1 mutants with specific PPPY motif alterations.
- SMURF-1 enhanced the release of wt and PPPY mutant HTLV-1 particles, but these virions exhibited abnormal morphology and reduced infectivity.
- Increased Gag ubiquitination correlated with budding defects induced by ITCH and SMURF-1 overexpression.
- Silencing of ITCH, but not other tested ubiquitin ligases, reduced HTLV-1 budding in 293T cells.
- Binding affinities between HTLV-1 LD motifs and WW domains did not correlate with the observed effects on viral budding.
Conclusions:
- ITCH plays a critical role in regulating HTLV-1 budding, primarily by modulating Gag ubiquitination.
- SMURF-1 also influences HTLV-1 release, but its effects lead to the production of non-infectious, morphologically aberrant virions.
- The functional impact of Nedd4 family ubiquitin ligases on HTLV-1 budding is not solely determined by their binding affinity to the viral late domains.
Related Concept Videos
01:24T Cell Types and Functions
Th1 cells stimulate dendritic cells to express necessary co-stimulatory molecules on their surfaces for...
01:29Immune Response Against Viral Pathogens
NK Cells
NK cells are a crucial part of our innate immune system, acting as the first line of defense against viral infections. These cells can recognize and kill infected cells without prior exposure to the virus, effectively slowing down the spread of infection. Additionally, NK cells produce proinflammatory...
01:27Cytotoxic T Cells-mediated Immune Response
Immunological surveillance is the ability of immune cells to monitor and eliminate infected cells with intracellular pathogens, neoplastically transformed cells, and cells with non-self antigens. Cytotoxic T cells and NK...