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Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress.The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.

SUBMITTER: Bergson S

PROVIDER: S-EPMC4248887 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

Bergson Shir S Kalt Inna I Itzhak Inbal I Brulois Kevin F KF Jung Jae U JU Sarid Ronit R

Journal of virology 20140827 21

<h4>Unlabelled</h4>Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confir ...[more]

PMID: 25165104

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Project description:UnlabelledCapsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a β-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein.ImportanceSeveral capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gammaherpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types and in KSHV-infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hypermethylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation450K and MethylationEpic BeadChip arrays and identified panels of hyper- and hypomethylated cellular promoters in KSHV-infected cells. We combined our genome-wide methylation analysis with high-throughput RNA sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hypermethylation and upregulated genes with hypomethylation. In addition, we show that treating the cells with a demethylating agent leads to reexpression of these downregulated genes, indicating that, indeed, DNA methylation plays a role in the repression of these human genes. Comparison between de novo infection and PEL suggests that the virus induces initial hypermethylation followed by a slow increase in genome-wide hypomethylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.IMPORTANCE In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a few methylated promoters have been identified in KSHV-infected cells. Here, we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV-infected cells. We have identified many hyper- and hypomethylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive cells from uninfected cells and may serve as the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV-associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here, we show that treating PEL cells with a demethylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

Dataset Information

Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

Publications

Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

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