Abstract
Viral DNA genomes replicating in cells encounter a myriad of host factors that facilitate or hinder viral replication. Viral proteins expressed early during infection modulate host factors interacting with viral genomes, recruiting proteins to promote viral replication, and limiting access to antiviral repressors. Although some host factors manipulated by viruses have been identified, we have limited knowledge of pathways exploited during infection and how these differ between viruses. To identify cellular processes manipulated during viral replication, we defined proteomes associated with viral genomes during infection with adenovirus, herpes simplex virus and vaccinia virus. We compared enrichment of host factors between virus proteomes and confirmed association with viral genomes and replication compartments. Using adenovirus as an illustrative example, we uncovered host factors deactivated by early viral proteins, and identified a subgroup of nucleolar proteins that aid virus replication. Our data sets provide valuable resources of virus-host interactions that affect proteins on viral genomes.
Highlights
From the ‡Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; §Division of Protective Immunity and Division of Cancer Pathobiology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ¶Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; ʈDivision of Infectious Diseases, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; **Protein and Proteomics Core, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; ‡‡Division of Infectious Diseases, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; §§Epigenetics Program, Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
When infected cells were treated with a 15 min EdU pulse at the peak of viral DNA synthesis (Ad5 at 24 hpi, herpes simplex virus type 1 (HSV-1) at 8 hpi and vaccinia virus (VACV) at 6 hpi), we observed a high number of cells with strong EdU signal (Fig. 1B and supplemental Fig. S1)
Each of these viruses express an early viral protein that has single-stranded DNA binding activity and marks viral replication factories or compartments (VRCs) (DBP for adenovirus type 5 (Ad5), ICP8 for HSV-1, and I3 for VACV), and the EdU labeling in infected cells was predominantly colocalized with these viral replication proteins (Fig. 1B)
Summary
Ad5, adenovirus C type 5; E1, adenovirus early coding region 1; E4, adenovirus early coding region 4; HSV-1, herpes simplex virus type-1; VACV, vaccinia virus; VRCs, viral replication compartments; MOI, multiplicity of infection; hpi, hours post infection; EdU, 5-ethynyl-2Ј-deoxyuridine; BrdU, 5-bromo-2Јdeoxyuridine; DAPI, 4Ј,6-diamidino-2-phenylindole, dihydrochloride; CHX, cycloheximide; iPOND, isolation of proteins on nascent DNA; NHEJ, nonhomologous end joining; BER, base excision repair; NER, nucleotide excision repair; ICL, interstrand crosslink repair; HRR, homologous recombination repair; VRCs, viral replication centers; GO, gene ontology; KOAc, potassium acetate; DTT, dithiothreitol; iBAQ, intensity-based absolute quantification. Viral proteins expressed early during the infectious process frequently alter the cellular environment to promote viral replication by inactivating host factors and preventing their association with viral DNA in VRCs where they could limit gene transcription or replication of viral genomes. By comparing proteomes associated with replicated DNA from cells uninfected or infected with Ad5, HSV-1 and VACV, we provide a comprehensive description of host factors and cellular processes potentially exploited by each virus during infection. We combined relative abundance with the fold change between Ad5 and Host proteomes to reveal potential targets of early viral proteins This analysis showed that i) TFII-I, a cellular transcription regulator, is inactivated by early viral proteins, ii) DNA repair proteins such as SLX4 can associate with viral replication centers and promote viral DNA replication, and iii) several nucleolar proteins, including TCOFI, are recruited to VRCs to promote viral replication. Our unbiased proteomics approach generated comprehensive databases that represent new resources to probe virus-host interactions and a framework for understanding how viruses manipulate host factors to redirect cellular processes and promote viral replication
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