Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G2 Arrest and Highly Effective Evasion of Innate Immune Sensing.

BK polyomavirus (BKPyV) is a small DNA virus that establishes a life-long persistent infection in the urinary tract of most people. BKPyV is known to cause severe morbidity in renal transplant recipients and can lead to graft rejection. The simple 5.2-kbp double-stranded DNA (dsDNA) genome expresses just seven known proteins; thus, it relies heavily on the host machinery to replicate. How the host proteome changes over the course of infection is key to understanding this host-virus interplay. Here, for the first time quantitative temporal viromics has been used to quantify global changes in >9,000 host proteins in two types of primary human epithelial cells throughout 72 h of BKPyV infection. These data demonstrate the importance of cell cycle progression and pseudo-G2 arrest in effective BKPyV replication, along with a surprising lack of an innate immune response throughout the whole virus replication cycle. BKPyV thus evades pathogen recognition to prevent activation of innate immune responses in a sophisticated manner.IMPORTANCE BK polyomavirus can cause serious problems in immune-suppressed patients, in particular, kidney transplant recipients who can develop polyomavirus-associated kidney disease. In this work, we have used advanced proteomics techniques to determine the changes to protein expression caused by infection of two independent primary cell types of the human urinary tract (kidney and bladder) throughout the replication cycle of this virus. Our findings have uncovered new details of a specific form of cell cycle arrest caused by this virus, and, importantly, we have identified that this virus has a remarkable ability to evade detection by host cell defense systems. In addition, our data provide an important resource for the future study of kidney epithelial cells and their infection by urinary tract pathogens.

Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE.

The steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different subcellular niches upon perturbation of the subcellular environment. Differential localisation, that is a change in the steady-state subcellular location of a protein, provides a step towards mechanistic insight of subcellular protein dynamics. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we describe a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to several datasets recovers well-studied translocations. In an application to cytomegalovirus infection, we obtain insights into the rewiring of the host proteome. Integration of other high-throughput datasets allows us to provide the functional context of these data.

Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation.

Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells.

Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist.

Vaccinia virus (VACV) has numerous immune evasion strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor κB (NF-κB), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics to quantify ∼9,000 cellular proteins and ∼80% of viral proteins at seven time points throughout VACV infection. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during infection. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a protein-based temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of HDAC5. Our approach thus identifies both a host antiviral factor and a viral mechanism of innate immune evasion.