Functional proteomic atlas of HIV infection in primary human CD4+ T cells.

Adi Naamati,James C Williamson,Nicholas J Matheson,Sara Marelli,Paul J Lehner,Edward Jd Greenwood

doi:10.7554/elife.41431

Abstract

Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell.

Highlights

Remodelling of the host proteome during viral infection may reflect direct effects of viral proteins, secondary effects or cytopathicity accompanying viral replication, or host countermeasures such as the interferon (IFN) response
To engineer a single round HIV reporter virus encoding streptavidin-binding peptide (SBP)-D LNGFR, we considered three settings in the proviral construct: fused to the endogenous Env signal peptide; or as an additional cistron, downstream of nef and either a Porcine teschovirus-1 2A (P2A) peptide or internal ribosome entry site (IRES)
The HIV-antibody-free magnetic cell sorting of infected cells (AFMACS) virus described in this study allows routine isolation of HIV-infected cells subjected to an multiplicity of infection (MOI) 1, avoiding artefacts associated with high MOIs and facilitating experiments in primary cells, where high levels of infection are difficult to achieve in practice

Summary

Introduction

Remodelling of the host proteome during viral infection may reflect direct effects of viral proteins, secondary effects or cytopathicity accompanying viral replication, or host countermeasures such as the interferon (IFN) response. We sought to apply our temporal proteomic approach to HIV infection of primary human CD4+ T lymphocytes, the principle cell type infected in vivo, at an MOI 1 To this end, we have developed an HIV reporter virus encoding a cell surface streptavidin-binding affinity tag, allowing antibody-free magnetic cell sorting of infected cells (AFMACS) (Matheson et al, 2014) (Figure 1A). We have developed an HIV reporter virus encoding a cell surface streptavidin-binding affinity tag, allowing antibody-free magnetic cell sorting of infected cells (AFMACS) (Matheson et al, 2014) (Figure 1A) This system allows rapid, scalable, affinity purification of HIV-infected cells from mixed cultures, bypassing the need for high MOIs or fluorescence-activated cell sorting (FACS). We use this system to generate a detailed atlas of cellular protein dynamics in HIV-infected primary human CD4+ T cells, show how this resource can be used to identify novel cellular proteins regulated by HIV, and assign causality to individual HIV accessory proteins

Results

B AFMACS 48h post-infection

Discussion

A Vif-dependent changes

Materials and methods

Ethics statement