Vpx is Critical for SIVmne infection of pigtail macaques

Michael A. Belshan, Jason T. Kimata, Charles Brown, Xiaogang Cheng, Anna McCulley, Alison Larsen, Rajesh Thippeshappa, Vida Hodara, Luis Giavedoni, Vanessa Hirsch, Lee Ratner

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Background: Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.Results: An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.Conclusions: This work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.

Original languageEnglish
Article number32
JournalRetrovirology
Volume9
DOIs
StatePublished - Apr 24 2012

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Macaca nemestrina
Viral Proteins
Infection
Macrophages
Macaca
Viruses
Reverse Transcription
Lymphocytes
Virus Integration
Defective Viruses
Ubiquitin-Protein Ligases
Cell Nucleus Active Transport
Viremia
Intestinal Mucosa
Rectum
Proline
Alanine
In Situ Hybridization
Tyrosine
Spleen

All Science Journal Classification (ASJC) codes

  • Virology
  • Infectious Diseases

Cite this

Belshan, M. A., Kimata, J. T., Brown, C., Cheng, X., McCulley, A., Larsen, A., ... Ratner, L. (2012). Vpx is Critical for SIVmne infection of pigtail macaques. Retrovirology, 9, [32]. https://doi.org/10.1186/1742-4690-9-32

Vpx is Critical for SIVmne infection of pigtail macaques. / Belshan, Michael A.; Kimata, Jason T.; Brown, Charles; Cheng, Xiaogang; McCulley, Anna; Larsen, Alison; Thippeshappa, Rajesh; Hodara, Vida; Giavedoni, Luis; Hirsch, Vanessa; Ratner, Lee.

In: Retrovirology, Vol. 9, 32, 24.04.2012.

Research output: Contribution to journalArticle

Belshan, MA, Kimata, JT, Brown, C, Cheng, X, McCulley, A, Larsen, A, Thippeshappa, R, Hodara, V, Giavedoni, L, Hirsch, V & Ratner, L 2012, 'Vpx is Critical for SIVmne infection of pigtail macaques', Retrovirology, vol. 9, 32. https://doi.org/10.1186/1742-4690-9-32
Belshan MA, Kimata JT, Brown C, Cheng X, McCulley A, Larsen A et al. Vpx is Critical for SIVmne infection of pigtail macaques. Retrovirology. 2012 Apr 24;9. 32. https://doi.org/10.1186/1742-4690-9-32
Belshan, Michael A. ; Kimata, Jason T. ; Brown, Charles ; Cheng, Xiaogang ; McCulley, Anna ; Larsen, Alison ; Thippeshappa, Rajesh ; Hodara, Vida ; Giavedoni, Luis ; Hirsch, Vanessa ; Ratner, Lee. / Vpx is Critical for SIVmne infection of pigtail macaques. In: Retrovirology. 2012 ; Vol. 9.
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abstract = "Background: Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.Results: An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.Conclusions: This work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.",
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AU - Larsen, Alison

AU - Thippeshappa, Rajesh

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N2 - Background: Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.Results: An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.Conclusions: This work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.

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