Quantitative Temporal Viromics: An Approach to Investigate Host-Pathogen Interaction

Weekes, Michael P.; Tomašec, Peter; Huttlin, Edward L.; Fielding, Ceri Alan; Nusinow, David P.; Stanton, Richard J.; Wang, Edward Chung Yern; Aicheler, Rebecca; Murrell, Isa; Wilkinson, Gavin William Grahame; Lehner, Paul J.; Gygi, Steven P.

doi:10.1016/j.cell.2014.04.028

Cited by 383 publications

(593 citation statements)

References 205 publications

(114 reference statements)

Supporting

Mentioning

540

Contrasting

Order By: Relevance

“…This is in accordance with a recent proteomics study of HCMV-infected cells, indicating that pUL77 and pUL93 are late proteins expressed simultaneously (57). In our immunoblotting experiments, pUL93 was seen as a major band migrating with a molecular mass of 68 kDa and as a minor form of 48 kDa.…”

Section: Discussionsupporting

confidence: 93%

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Borst

Bauerfeind

Binz

et al. 2016

J Virol

View full text Add to dashboard Cite

Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.T he life cycle of human cytomegalovirus (HCMV), the prototype member of the betaherpesviruses, comprises a nuclear phase that includes transcription of viral genes, replication of the double-stranded DNA genome, assembly of procapsids, packaging of the viral DNA into the preformed capsids, and maturation of the DNA-filled c...

show abstract

Section: Discussionsupporting

confidence: 93%

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Borst

Bauerfeind

Binz

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…to be regulated efficiently during viral infection by a mechanism analogous to viral downregulation of MHC class I or NKG2D ligands, implying that immune evasion of BTN-mediated activation of γδ T cells is a requirement for viral infection (76)(77)(78).…”

Section: Mr1-restricted Presentation Of Bacterial Metabolites To Maitmentioning

confidence: 99%

Regulation of Immunity by Butyrophilins

Rhodes

Reith

Trowsdale

2016

Annu. Rev. Immunol.

129

132

View full text Add to dashboard Cite

Butyrophilin molecules (commonly contracted to BTN), collectively take their name from the eponymous protein in cow's milk. They are considered to be members of the B7 family of costimulatory receptors, which includes B7.1 (CD80), B7.2 (CD86), and related molecules, such as PD-L1 (B7-H1, CD274), ICOS-L (CD275), and B7-H3 (CD276). These coreceptors modulate T cell responses upon antigen presentation by major histocompatibility complex and cognate αβ T cell receptor engagement. Molecules such as BTN3A1 (CD277), myelin oligodendrocyte glycoprotein, and mouse Skint1 and Btnl2, all members of the butyrophilin family, show greater structural and functional diversity than the canonical B7 receptors. Some butyrophilins mediate complex interactions between antigen-presenting cells and conventional αβ T cells, and others regulate the immune responses of specific γδ T cell subsets by mechanisms that have characteristics of both innate and adaptive immunity.

show abstract

“…Furthermore, several of the targets of siRNA in our screening data were not found in a proteomic study of HFFs infected with HCMV (27). Therefore, analysis of siRNA off-target binding alone may not be sufficient to exclude siRNAs from analysis.…”

Section: Discussionmentioning

confidence: 90%

“…Indeed, analysis of siRNA screening data can include a comparison of screening hits with gene expression profiles to identify false positives (7). Further analysis of siRNA screening data for HCMV-infected cells should benefit from a comparison of siRNA screening hits with proteins known to be expressed in HCMVinfected cells (27), as we have performed here. It remains unknown what RNAs are targeted by the siRNAs that we judged to be false positives in our screening data.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

Polachek

Moshrif

Franti

et al. 2016

J Virol

View full text Add to dashboard Cite

High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCEThere is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell. I dentification of factors encoded by the cell that are required for virus replication can illuminate important features of virus-host interactions and identify novel drug targets for therapeutic intervention. Stages of productive human cytomegalovirus (HCMV) replication take place in both the nucleus and the cytoplasm (1). After replication of the viral DNA genome in the nucleus, newly synthesized viral genomic DNA is packaged into nascent capsids in the nucleus. These capsids then bud through the nuclear membrane and, after accessing a viral assembly compartment in the cytoplasm (2), undergo a process of secondary envelopment in the cytoplasm in which capsids gain a lipid bilayer before exiting the cell (1). Many of these processes require the function of cellular factors, a number of which are unknown.High-throughput small interfering RNA (siRNA) screening has been a successful strategy to identify cellular factor...

show abstract

Quantitative Temporal Viromics: An Approach to Investigate Host-Pathogen Interaction

Cited by 383 publications

References 205 publications

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Regulation of Immunity by Butyrophilins

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

Contact Info

Product

Resources

About