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Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Pieter Meuwissen UGent, Bettina Stolp, Veronica Iannucci, JOLIEN VERMEIRE, Evelien Naessens UGent, Kalle Saksela, Matthias Geyer, Guido Vanham, Kevin Ariën UGent, Oliver T Fackler, et al. (2012) RETROVIROLOGY. 9.
abstract
BACKGROUND: The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. RESULTS: The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef's association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. CONCLUSION: Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
MAJOR CORECEPTOR, HIV, PRIMATE LENTIVIRUSES, IMMUNOLOGICAL SYNAPSE, TYPE-1 NEF, MHC-I, T-CELL DEVELOPMENT, AIDS-LIKE DISEASE, I DOWN-REGULATION, TRANSGENIC MIC, Receptor downregulation, SH3 domain binding, Nef, Sequence motifs, Infectivity, Replication, HUMAN-IMMUNODEFICIENCY-VIRUS, Cytoskeleton, Lck
journal title
RETROVIROLOGY
Retrovirology
volume
9
article number
34
pages
19 pages
Web of Science type
Article
Web of Science id
000307821200001
JCR category
VIROLOGY
JCR impact factor
5.657 (2012)
JCR rank
5/34 (2012)
JCR quartile
1 (2012)
ISSN
1742-4690
DOI
10.1186/1742-4690-9-34
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
3130578
handle
http://hdl.handle.net/1854/LU-3130578
date created
2013-02-14 10:40:57
date last changed
2016-12-21 15:42:09
@article{3130578,
  abstract     = {BACKGROUND: The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region.
RESULTS: The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF{\textrightarrow}AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef's association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck.
CONCLUSION: Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.},
  articleno    = {34},
  author       = {Meuwissen, Pieter and Stolp, Bettina and Iannucci, Veronica and VERMEIRE, JOLIEN and Naessens, Evelien and Saksela, Kalle and Geyer, Matthias and Vanham, Guido and Ari{\"e}n, Kevin and Fackler, Oliver T and Verhasselt, Bruno},
  issn         = {1742-4690},
  journal      = {RETROVIROLOGY},
  keyword      = {MAJOR CORECEPTOR,HIV,PRIMATE LENTIVIRUSES,IMMUNOLOGICAL SYNAPSE,TYPE-1 NEF,MHC-I,T-CELL DEVELOPMENT,AIDS-LIKE DISEASE,I DOWN-REGULATION,TRANSGENIC MIC,Receptor downregulation,SH3 domain binding,Nef,Sequence motifs,Infectivity,Replication,HUMAN-IMMUNODEFICIENCY-VIRUS,Cytoskeleton,Lck},
  language     = {eng},
  pages        = {19},
  title        = {Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function},
  url          = {http://dx.doi.org/10.1186/1742-4690-9-34},
  volume       = {9},
  year         = {2012},
}

Chicago
Meuwissen, Pieter, Bettina Stolp, Veronica Iannucci, Jolien Vermeire, Evelien Naessens, Kalle Saksela, Matthias Geyer, et al. 2012. “Identification of a Highly Conserved Valine-glycine-phenylalanine Amino Acid Triplet Required for HIV-1 Nef Function.” Retrovirology 9.
APA
Meuwissen, P., Stolp, B., Iannucci, V., Vermeire, J., Naessens, E., Saksela, K., Geyer, M., et al. (2012). Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function. RETROVIROLOGY, 9.
Vancouver
1.
Meuwissen P, Stolp B, Iannucci V, Vermeire J, Naessens E, Saksela K, et al. Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function. RETROVIROLOGY. 2012;9.
MLA
Meuwissen, Pieter, Bettina Stolp, Veronica Iannucci, et al. “Identification of a Highly Conserved Valine-glycine-phenylalanine Amino Acid Triplet Required for HIV-1 Nef Function.” RETROVIROLOGY 9 (2012): n. pag. Print.