Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation.
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ABSTRACT:
SUBMITTER: Gabaev I
PROVIDER: S-EPMC7567700 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Kaposi’s Sarcoma associated herpesvirus (KSHV) is an oncogenic human virus and leading cause of mortality in HIV infection. Reactivation of KSHV from latent to lytic stage infection initiates a cascade of viral gene expression, and here we show how these changes remodel the host cell proteome to enable viral replication. By undertaking a systematic and unbiased analysis of changes to the endothelial cell proteome following lytic KSHV reactivation, we quantify >7000 cellular and 71 viral proteins. Lytic KSHV infection resulted in >2-fold downregulation of 291 cellular proteins, including PKR, the key cellular sensor of double-stranded RNA. A complementary KSHV genome-wide CRISPR genetic screen identified K5 as the viral gene responsible for the downregulation of two novel KSHV targets, Nectin-2 and CD155, both ligands of the NK cell DNAM-1 receptor. Despite the high episome copy number, we show that CRISPR Cas9 provides a remarkably efficient way to target KSHV genomes.
2021-08-23 | PXD021387 | Pride
Project description:Kaposi’s Sarcoma associated herpesvirus (KSHV) is an oncogenic human virus and leading cause of mortality in HIV infection. Reactivation of KSHV from latent to lytic stage infection initiates a cascade of viral gene expression, and here we show how these changes remodel the host cell proteome to enable viral replication. By undertaking a systematic and unbiased analysis of changes to the endothelial cell proteome following lytic KSHV reactivation, we quantify >7000 cellular and 71 viral proteins. Lytic KSHV infection resulted in >2-fold downregulation of 291 cellular proteins, including PKR, the key cellular sensor of double-stranded RNA. A complementary KSHV genome-wide CRISPR genetic screen identified K5 as the viral gene responsible for the downregulation of two novel KSHV targets, Nectin-2 and CD155, both ligands of the NK cell DNAM-1 receptor. Despite the high episome copy number, we show that CRISPR Cas9 provides a remarkably efficient way to target KSHV genomes.
2022-05-26 | PXD017365 | Pride
Project description:Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.
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Project description:Target identification of small molecules is a great challenge but an essential step in drug discovery. Here, a quantitative proteomics approach has been used to characterize the cellular targets of DR, a DDR1 inhibitor. By taking advantage of competitive affinity-based protein profiling coupled with bioimaging, Cathepsin D (CTSD) was found to be the principle off-target of DR in human cancer cells. Further findings suggest the potential of DR as a novel CTSD inhibitor for breast cancer treatment. In addition, a trans-cyclooctene (TCO) containing probe was developed to track the binding between DR and its target proteins in living systems and could be a useful tool for DDR1 detection.
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Project description:RNA polyadenylation (pA) is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV) infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ~90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site) in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3'RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A) signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6), K9 (vIRF1), K10.5 (vIRF3), K11 (vIRF2), K12 (Kaposin A), T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression.
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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman's diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development.IMPORTANCEThe reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases.
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