Expression Profiling of Herpesvirus and Vaccinia Virus Proteins Using a High-Throughput Baculovirus Screening System

Gao, Mian; Brufatto, Nicole; Chen, Tricia; Murley, Laura Lea; Thalakada, Rosanne; Domagala, Megan; Beattie, Bryan K.; Mamelak, Daniel; Athanasopoulos, Vicki; Johnson, David C.; McFadden, Grant; Burks, Christian; Frappier, Lori

doi:10.1021/pr050137u

Cited by 15 publications

(10 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is possible some vaccinia proteins were poorly expressed in our E. coli library. Gao et al (81) used a different fusion partner and Coomassie blue staining, an insensitive method, and found 16% of 50 full-length vaccinia ORFs were not expressed. One of their nonexpressed ORFs, E4L, was definitely expressed by our library (Tables II and III).…”

Section: Discussionmentioning

confidence: 99%

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

Jing¹,

Chong

Byrd³

et al. 2007

The Journal of Immunology

View full text Add to dashboard Cite

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.

show abstract

Section: Discussionmentioning

confidence: 99%

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

Jing¹,

Chong

Byrd³

et al. 2007

The Journal of Immunology

View full text Add to dashboard Cite

show abstract

“…Expression libraries for HSV-1 (strain 17), CMV (strain AD169) and EBV (strain B95-8) were generated by PCR amplification of each predicted open reading frame (ORF), according to the NCBI entries for each virus, from viral genomic DNA samples generated as described in [75]. Nineteen additional CMV ORFs (UL133-151) not present in AD169 were obtained by PCR amplification from the large Towne strain of CMV.…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies

Salsman

Zimmerman

Chen³

et al. 2008

PLoS Pathog

Self Cite

122

View full text Add to dashboard Cite

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.

show abstract

“…A high-throughput method for baculovirus expression in 24-well blocks was developed, as was a high-throughput procedure for protein purification, and proteins were then assayed for solubility. Of 78 VACV proteins tested, 62 genes (79%) were expressed in this system, and this will allow the production of antibodies against these proteins and more detailed physical studies (60).…”

Section: Global Protein Expression Studiesmentioning

confidence: 99%

Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

Self Cite

View full text Add to dashboard Cite

SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.

show abstract

Expression Profiling of Herpesvirus and Vaccinia Virus Proteins Using a High-Throughput Baculovirus Screening System

Cited by 15 publications

References 3 publications

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies

Poxvirus Proteomics and Virus-Host Protein Interactions

Contact Info

Product

Resources

About