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Human immunodeficiency virus matrix tyrosine phosphorylation: characterization of the kinase and its substrate
D Camaur1, P Gallay, S Swingler
1Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Abstract:
During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated.
Insights
A cellular protein kinase phosphorylates human immunodeficiency virus (HIV) matrix (MA) on tyrosine, aiding viral infection. This kinase activity is found in various cells and membranes, and doesn't require a specific MA sequence.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Phosphorylation of human immunodeficiency virus (HIV) matrix (MA) on C-terminal tyrosine is crucial for infecting non-dividing cells.
- This modification facilitates the import of MA into the viral core and preintegration complex.
- A cellular protein kinase, incorporated into virions, mediates this essential MA tyrosine phosphorylation.
Purpose of the Study:
- To investigate the nature of the cellular protein kinase responsible for MA tyrosine phosphorylation.
- To identify the determinants within MA necessary for its phosphorylation by this kinase.
Main Methods:
- In vitro kinase assays were used to detect MA tyrosine kinase activity in various cell lines and primary human cells.
- Cell fractionation was employed to determine the subcellular localization of the kinase activity.
- Mutagenesis of the C-terminal residues of HIV-1 MA was performed to assess their role in phosphorylation.
- MA from murine leukemia virus was analyzed for phosphorylation.
Main Results:
- MA tyrosine kinase activity was detected in T-lymphoid cells, B cells, kidney fibroblasts, HeLa cells, platelets, macrophages, and activated peripheral blood lymphocytes (PBLs).
- The kinase activity was not found in erythrocytes or resting PBLs.
- Subcellular localization studies revealed the kinase activity is enriched in cellular membranes.
- The kinase in HIV-2 particles associates with the inner viral membrane leaflet, while phosphorylated MA is in the core.
- Mutations in the C-terminal residues of HIV-1 MA did not prevent phosphorylation.
- Murine leukemia virus MA was also found to be C-terminally tyrosine phosphorylated.
Conclusions:
- The cellular protein kinase responsible for HIV MA tyrosine phosphorylation is widely distributed in various cell types and is membrane-associated.
- The phosphorylation does not require a highly specific sequence adjacent to the C-terminal tyrosine residue of MA.
- This phosphorylation mechanism appears conserved across different retroviruses, as evidenced by murine leukemia virus MA phosphorylation.