Singh S P, Gupta P, Mahalingam S
HIV-2/SIV Vpx Protein Interacts with Human Nup153 and Regulates Viral Pathogenesis Singh S P, Gupta P, Mahalingam S Laboratory of Molecular Virology and Cell Biology, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India. Email: [email protected] or [email protected]
Abstract
Results
Vpx, a virus associated accessory protein is encoded by Human Immunodeficiency Virus
Vpr of HIV-1 and Vpx of SIVsmPBj interacts with endogenous Nup153
Ser and Tyr residues of Vpx are important for its Nup153 interaction
ERK-2 inhibitor abolishes the interaction between Vpx and Nup153
type 2 (HIV-2) and Simian Immunodeficiency Virus (SIVsm/SIVmac lineage) known to be involved in nuclear import of viral DNA. Non-dividing cells such as macrophages are the primary targets of lentiviruses. Once virus enters into cytoplasm, uncoating of capsid occurs and viral cDNA is completed with the help of viral reverse transcriptase. cDNA
along with other viral and host proteins comprise the pre-integration complex (PIC) which is translocated to the nucleus for viral genome integration into host chromosome. Vpx
Figure 7. Co-immunoprecipitation of Vpx with endogenous Nup153 in presence of
being an integral part of PIC was found to be critical for viral DNA nuclear import as Vpx
ERK-2 inhibitors U0126 and Hypericin. Figure 1. A.
mutant virus failed to replicate in non-dividing cells. The exact mechanism by which Vpx
Nup153
Co-immunoprecipitation of
with
GFP-tagged
Vpr/Vpx
helps in this virus replication remains unknown. Molecular transport across nuclear
proteins. B. Co-localization of GFP-tagged
envelope is governed by nucleopore complexes (NPCs), composed of proteins called
Vpr/Vpx and Nup153 proteins.
Figure 4. A. Co-immunoprecipitation of GFP-tagged Vpx mutant proteins with endogenous Nup153. B. Line diagram of Vpx point mutant constructs.
Vpx mutant viruses are defective in viral DNA nuclear import ability
Variable localization of Nup153 interaction defective Vpx proteins
nucleoporins (NUPs). Our data suggests that Vpx interaction with Nup153 is critical for Vpx interacts with Zinc finger domain of Nup153
nuclear import of viral genome.
Figure 8. 2-LTR assay of wild type Vpx mutant SIVsmPBj viruses.
Materials and Methods Conclusions
Construction of Vpx and Nup153 expression plasmids: Vpx full length and deletions constructs were cloned between EcoRI and XhoI sites of pCDNA3.1 as N-GFP or N-Flag
• HIV/SIV Vpx co-localize and interacts with Nup153.
fusion. Similarly Nup153 deletion constructs were cloned in pCDNA3.1 N-HA fusion.
• Zinc Finger domain (610-869 aa) of Nup153 was found to be critical for its interaction with Vpx.
Co-Immunoprecipitation: Wild type and mutant Vpx proteins were overexpressed in 293T cells and pre-cleared cell lysates were used for immunoprecipitation using anti
Figure 5. A. Localization of GFP-tagged Vpx wild type and mutant proteins. B.
Nup153 or anti GFP/Flag antibody and immunopreciptaed proteins were analyzed by
Figure 2. A. Co-immunoprecipitation of HA-tagged Nup153 deletion constructs with
western blotting.
GFP-tagged Vpx. B. Schematic representation of Nup153 domains. C. Docking
Immunostaining: HeLa cells were seeded in a four well chambered slide and transfected
analysis Vpx interaction with Zinc finger domain of NUp153.
with GFP-tagged Vpx/Vpr expression plasmids. Immunostaining was done using anti
Structure of wild type and mutant Vpx proteins.
Nup153 interacts variably with Vpx from different HIV/SIVs
• Exchange of serine (63,65) and tyrosine (66, 69 and 71) residues
impaired Vpx interaction with Nup153. • Vpx from different HIV/SIV viruses interact variably with Nup153 and presence of ERK-2 inhibitor abolishes Vpx interaction with Nup153. These findings suggests importance of ERK-2 mediated Vpx phosphorylation.
C-terminal domain of Vpx interacts with Nup153
• 2-LTR assay showed defective nuclear import ability of Vpx mutant
Nup153 antibody and images were captured using confocal microscopy.
viruses.
Virus stock and ELISA: Full length molecular clones of wild type and mutant SIVsmPBj viruses were transfected in 293T cells and supernatants were collected and virus titer was determined by p27 antigen capture assay.
Acknowledgement
2-LTR assay: Cell cycle arrest at G2/M was obtained by treating CEMX174 with 1 µM
Department of Biotechnology, (Grant No: BT/PR8142/MED/29/709/2013) for
Etoposide for 24 h. Genomic DNA was isolated 6 h and 12 h post transduction and 2-LTR
funding and Ministry of Human Resource Development for scholarship. NIH
junction sequence was PCR amplified using U5 and U3 specific primers. Beta actin PCR
AIDS Reagent Program for providing antibodies and cell lines. Dr. Katharine
was done as a control. p27(110-165) region was PCR amplified for total viral DNA content.
Figure 3. A. Schematic representation of GFP-tagged deletion constructs of Vpx. B. Co-immunoprecipitation of GFP-tagged Vpx deletion constructs with endogenous
Figure 6. A. Co-immunoprecipitation of endogenous Nup153 with GFP tagged Vpx
S. Ullman, University of Utah, for providing GFP-Nup153 expression
Nup153.
from different HIV/SIVs. B. Line diagram of different Vpx proteins.
plasmid.
Abstract
Results
Vpx, a virus associated accessory protein is encoded by Human Immunodeficiency Virus
Vpr of HIV-1 and Vpx of SIVsmPBj interacts with endogenous Nup153
Ser and Tyr residues of Vpx are important for its Nup153 interaction
ERK-2 inhibitor abolishes the interaction between Vpx and Nup153
type 2 (HIV-2) and Simian Immunodeficiency Virus (SIVsm/SIVmac lineage) known to be involved in nuclear import of viral DNA. Non-dividing cells such as macrophages are the primary targets of lentiviruses. Once virus enters into cytoplasm, uncoating of capsid occurs and viral cDNA is completed with the help of viral reverse transcriptase. cDNA
along with other viral and host proteins comprise the pre-integration complex (PIC) which is translocated to the nucleus for viral genome integration into host chromosome. Vpx
Figure 7. Co-immunoprecipitation of Vpx with endogenous Nup153 in presence of
being an integral part of PIC was found to be critical for viral DNA nuclear import as Vpx
ERK-2 inhibitors U0126 and Hypericin. Figure 1. A.
mutant virus failed to replicate in non-dividing cells. The exact mechanism by which Vpx
Nup153
Co-immunoprecipitation of
with
GFP-tagged
Vpr/Vpx
helps in this virus replication remains unknown. Molecular transport across nuclear
proteins. B. Co-localization of GFP-tagged
envelope is governed by nucleopore complexes (NPCs), composed of proteins called
Vpr/Vpx and Nup153 proteins.
Figure 4. A. Co-immunoprecipitation of GFP-tagged Vpx mutant proteins with endogenous Nup153. B. Line diagram of Vpx point mutant constructs.
Vpx mutant viruses are defective in viral DNA nuclear import ability
Variable localization of Nup153 interaction defective Vpx proteins
nucleoporins (NUPs). Our data suggests that Vpx interaction with Nup153 is critical for Vpx interacts with Zinc finger domain of Nup153
nuclear import of viral genome.
Figure 8. 2-LTR assay of wild type Vpx mutant SIVsmPBj viruses.
Materials and Methods Conclusions
Construction of Vpx and Nup153 expression plasmids: Vpx full length and deletions constructs were cloned between EcoRI and XhoI sites of pCDNA3.1 as N-GFP or N-Flag
• HIV/SIV Vpx co-localize and interacts with Nup153.
fusion. Similarly Nup153 deletion constructs were cloned in pCDNA3.1 N-HA fusion.
• Zinc Finger domain (610-869 aa) of Nup153 was found to be critical for its interaction with Vpx.
Co-Immunoprecipitation: Wild type and mutant Vpx proteins were overexpressed in 293T cells and pre-cleared cell lysates were used for immunoprecipitation using anti
Figure 5. A. Localization of GFP-tagged Vpx wild type and mutant proteins. B.
Nup153 or anti GFP/Flag antibody and immunopreciptaed proteins were analyzed by
Figure 2. A. Co-immunoprecipitation of HA-tagged Nup153 deletion constructs with
western blotting.
GFP-tagged Vpx. B. Schematic representation of Nup153 domains. C. Docking
Immunostaining: HeLa cells were seeded in a four well chambered slide and transfected
analysis Vpx interaction with Zinc finger domain of NUp153.
with GFP-tagged Vpx/Vpr expression plasmids. Immunostaining was done using anti
Structure of wild type and mutant Vpx proteins.
Nup153 interacts variably with Vpx from different HIV/SIVs
• Exchange of serine (63,65) and tyrosine (66, 69 and 71) residues
impaired Vpx interaction with Nup153. • Vpx from different HIV/SIV viruses interact variably with Nup153 and presence of ERK-2 inhibitor abolishes Vpx interaction with Nup153. These findings suggests importance of ERK-2 mediated Vpx phosphorylation.
C-terminal domain of Vpx interacts with Nup153
• 2-LTR assay showed defective nuclear import ability of Vpx mutant
Nup153 antibody and images were captured using confocal microscopy.
viruses.
Virus stock and ELISA: Full length molecular clones of wild type and mutant SIVsmPBj viruses were transfected in 293T cells and supernatants were collected and virus titer was determined by p27 antigen capture assay.
Acknowledgement
2-LTR assay: Cell cycle arrest at G2/M was obtained by treating CEMX174 with 1 µM
Department of Biotechnology, (Grant No: BT/PR8142/MED/29/709/2013) for
Etoposide for 24 h. Genomic DNA was isolated 6 h and 12 h post transduction and 2-LTR
funding and Ministry of Human Resource Development for scholarship. NIH
junction sequence was PCR amplified using U5 and U3 specific primers. Beta actin PCR
AIDS Reagent Program for providing antibodies and cell lines. Dr. Katharine
was done as a control. p27(110-165) region was PCR amplified for total viral DNA content.
Figure 3. A. Schematic representation of GFP-tagged deletion constructs of Vpx. B. Co-immunoprecipitation of GFP-tagged Vpx deletion constructs with endogenous
Figure 6. A. Co-immunoprecipitation of endogenous Nup153 with GFP tagged Vpx
S. Ullman, University of Utah, for providing GFP-Nup153 expression
Nup153.
from different HIV/SIVs. B. Line diagram of different Vpx proteins.
plasmid.
