Proteomic Analyses of Human Cytomegalovirus Strain AD169 Derivatives Reveal Highly Conserved Patterns of Viral and Cellular Proteins in Infected Fibroblasts

Sabine Reyda,Nicole Büscher,Bodo Plachter,Stefan Tenzer

doi:10.3390/v6010172

Abstract

Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.

Highlights

The human cytomegalovirus (HCMV) is a pathogen of substantial clinical relevance that may lead to severe disease and sequelae after prenatal infection and life threating conditions in immunosuppressed individuals
It was yet remarkable that viral proteins could be detected at this early time after infection, underscoring the sensitivity of the proteomic approach
AD169 descendants is subject to only subtle variations. This argues in favor of a conserved process of viral protein packaging within a given strain of Human cytomegalovirus (HCMV)

Summary

Introduction

The human cytomegalovirus (HCMV) is a pathogen of substantial clinical relevance that may lead to severe disease and sequelae after prenatal infection and life threating conditions in immunosuppressed individuals. Tegument attachment to the capsids occurs predominantly at the AC and precedes envelopment Both virions and subviral dense bodies are enveloped at AC and are subsequently found in cytoplasmic vesicles that are transported to the cell surface by an exocytotic pathway [6,13]. Analyses of the protein composition of HCMV virions by mass spectrometry were focused on one single AD169-derivative. Up to this point, it remained unclear how culturing of related HCMVs in different laboratories would influence the proteomes of infected cells and virions. The pp65neg strain RV-KB14 was generated by inserting a tetracycline resistance cassette into the UL83 (pp65) ORF of pAD/cre (the BAC clone used for reconstitution of RV-BADwt), thereby deleting all of the pp65-coding region except for 152 5'. No gross alteration of the viral protein content of the virions of pp65pos- or pp65neg-viruses were seen

Conserved Pattern of Viral Protein Expression in Infected Cells

Comparable Impact of RV-HB15- and RV-BADwt-Infection on the Cellular Proteome

Conserved Stoichiometry of Viral Proteins in Virions of AD169-Derived Viruses

Experimental Section

Conclusions