GSBS Dissertations and Theses

Approval Date

12-14-2009

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Immunology and Virology

Subjects

Viral Regulatory and Accessory Proteins; vpr Gene Products, Human Immunodeficiency Virus; Simian immunodeficiency virus; Lentiviruses, Primate; Dissertations, UMMS; Academic Dissertations

Abstract

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/ Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. The functions of Vpx/ Vpr are not well understood. This study presents evidence that the Vpx proteins of HIV-2/ SIVSM promote HIV-1 infection by antagonizing an antiviral restriction in myeloid cells.

Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Similarly, the cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx rendered macrophages permissive to MLV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr.

This study further demonstrates that this restriction prevents transduction of quiescent monocytes by HIV-1. Although terminally differentiated macrophages are partially permissive to HIV-1, quiescent monocytes, which are macrophage precursors, are highly refractory to lentiviral infection. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection, suggesting the resistance of quiescent monocytes to HIV-1 transduction is governed by a restriction factor. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Introduction of Vpx into monocytes by pre-infection also rendered quiescent monocytes permissive to HIV-1 infection. Infection of monocytes by HIV-1 either with or without Vpx did not have an effect on temporal expression of CD71. In addition, Vpx increased permissivity of CD71 and CD71+ cells to HIV-1 infection with no apparent bias. These results confirm that Vpx directly renders undifferentiated monocytes permissive to HIV-1 transduction without inducing their differentiation. The introduction of Vpx did not significantly alter APOBEC3G complex distribution, suggesting a restriction other than APOBEC3G was responsible for the resistance of monocytes to HIV-1.

Collectively our results indicate that macrophages and monocytes harbor a potent antiviral restriction that is counteracted by the Vpx protein. The relative ability of primate lentiviruses and gammaretroviruses to transduce non-dividing myeloid-cells is dependent upon their ability to neutralize this restriction.

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.