scholarly journals A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

2021 ◽  
Author(s):  
Mariel Kleer ◽  
Grant MacNeil ◽  
Eric S. Pringle ◽  
Jennifer A. Corcoran
Keyword(s):  
Kaposi's Sarcoma ◽  
De Novo ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Nuclear Bodies ◽  
Processing Bodies ◽  
Recombinant Viruses ◽  
Precise Analysis ◽  
Genome Copy Number

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several human cancers including the endothelial cell (EC) malignancy, Kaposi's sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, known as K-genes, are typically important for KSHV replication and pathogenesis. Among the K-genes, the kaposin mRNA is highly expressed in both latent and lytic phases of infection, but its polycistronic nature has hindered methodical analysis of the role of kaposin translation products in viral replication. At least three proteins are produced from the kaposin transcript, Kaposin A (KapA), B (KapB), and C (KapC). We have previously shown that KapB overexpression is sufficient to recapitulate two KS phenotypes, EC spindling and elevated proinflammatory cytokine transcripts, the latter which proceeds via the disassembly of RNA decay granules called processing bodies (PBs). To pinpoint the relative contributions of kaposin proteins at different stages of KSHV infection, we constructed four recombinant viruses by deleting or recoding the kaposin locus. Latent infection of iSLK cells with kaposin-deficient viruses resulted in reduced viral genome copy number and small LANA nuclear bodies; despite this, all iSLK cells were capable of progeny virion production. De novo infection of ECs revealed that KapB was dispensable for EC spindling but required for PB disassembly during KSHV latency, suggesting other viral proteins contribute to spindling. These findings demonstrate that our panel of viruses enables precise analysis of respective contributions of individual kaposin proteins to KSHV replication. This approach serves as a guide for the functional analysis of complex multicistronic viral loci.

scholarly journals The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

2016 ◽  
Vol 90 (17) ◽  
pp. 7657-7666 ◽  
Author(s):  
Zhigang Zhang ◽  
Wuguo Chen ◽  
Marcia K. Sanders ◽  
Kevin F. Brulois ◽  
Dirk P. Dittmer ◽  
...  
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Akt Kinase ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOPdisplayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REVupon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOPalso yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.

New Insights into the Viral Theory of Amyotrophic Lateral Sclerosis: Study on the Possible Role of Kaposi’s Sarcoma-Associated Virus/Human Herpesvirus 8

2002 ◽  
Vol 47 (2) ◽  
pp. 108-112 ◽  
Author(s):  
Patrizia Sola ◽  
Roberta Bedin ◽  
Federica Casoni ◽  
Patrizia Barozzi ◽  
Jessica Mandrioli ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Lateral Sclerosis

Kaposi's sarcoma, human herpesvirus 8 infection and the potential role of promoter-arthropod bites in northern Sweden

2006 ◽  
Vol 78 (11) ◽  
pp. 1452-1455 ◽  
Author(s):  
Valeria Ascoli ◽  
Luca Facchinelli ◽  
Laura Valerio ◽  
Daniela Manno ◽  
Mario Coluzzi
Keyword(s):  
Kaposi's Sarcoma ◽  
Potential Role ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Northern Sweden ◽  
Herpesvirus 8

scholarly journals A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

2021 ◽  
Author(s):  
Mariel Kleer ◽  
Grant MacNeil ◽  
Nancy Adam ◽  
Eric S. Pringle ◽  
Jennifer A. Corcoran
Keyword(s):  
Functional Analysis ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Kshv Infection ◽  
Rna Granules ◽  
Precise Analysis ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposi’s sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the “K-genes”, are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. In previous work, we showed KapB alone recapitulated the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). Using the new ΔKapB virus, we showed that KapB was necessary for this effect during latent KSHV infection. Moreover, we observed that despite the ability of all kaposin-deficient latent iSLK cell lines to produce virions, all displayed low viral episome copy number, a defect that became more pronounced after primary infection of naïve ECs. For ΔKapB, provision of KapB in trans failed to complement the defect, suggesting a requirement for the kaposin locus in cis . These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci. Importance Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.

scholarly journals Carboxy Terminus of Human Herpesvirus 8 Latency-Associated Nuclear Antigen Mediates Dimerization, Transcriptional Repression, and Targeting to Nuclear Bodies

2000 ◽  
Vol 74 (18) ◽  
pp. 8532-8540 ◽  
Author(s):  
David R. Schwam ◽  
Randy L. Luciano ◽  
Shahana S. Mahajan ◽  
LaiYee Wong ◽  
Angus C. Wilson
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Nuclear Bodies ◽  
Human Herpesvirus 8 ◽  
Nuclear Antigen ◽  
Intact Protein ◽  
Viral Gene ◽  
Herpesvirus 8 ◽  
C Terminus

ABSTRACT Human herpesvirus 8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus) is the causative agent of Kaposi's sarcoma and certain B-cell lymphomas. In most infected cells, HHV-8 establishes a latent infection characterized by the expression of latency-associated nuclear antigen (LANA) encoded by open reading frame 73. Although unrelated by sequence, there are functional similarities between LANA and the EBNA-1 protein of Epstein-Barr virus. Both accumulate as subnuclear speckles and are required for maintenance of the viral episome. EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection. Here we show that LANA forms stable dimers, or possibly higher-order multimers, and that this is mediated by a conserved region in the C terminus. By expressing a series of truncations, we show that both the N- and C-terminal regions localize to the nucleus, although only the C terminus accumulates as nuclear speckles characteristic of the intact protein. Lastly, we show that LANA can function as a potent transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. Domains in both the N and C termini mediate repression. This suggests that one function of LANA is to suppress the expression of the viral lytic genes or cellular genes involved in the antiviral response.

scholarly journals Latent nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 induces and relocates RING3 to nuclear heterochromatin regions

2002 ◽  
Vol 83 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Karin Mattsson ◽  
Csaba Kiss ◽  
Georgina M. Platt ◽  
Guy R. Simpson ◽  
Elena Kashuba ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Body Cavity ◽  
Nuclear Bodies ◽  
Human Herpesvirus 8 ◽  
Nuclear Antigen ◽  
Lymphoma Cells ◽  
Herpesvirus 8 ◽  
C Terminus

LANA, the major latency-associated nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV-8), binds RING3 protein, one of five human homologues of the fsh (female sterile homeotic) gene product of Drosophila. In KSHV/HHV-8-infected cells LANA and the viral episomes accumulate in heterochromatin-associated nuclear bodies. Here we show that in several KSHV/HHV-8-negative cell lines derived from carcinomas, sarcomas and lymphomas, RING3 was expressed at low levels, primarily localized to the euchromatin, and dissociated from the chromosomes during mitosis. In contrast, in KSHV/HHV-8-infected body cavity lymphoma cells the bulk of RING3 localizes to the LANA nuclear bodies and remains associated with the chromosomes during cell division. KSHV/HHV-8-infected body cavity lymphoma cells expressed RING3 at much higher levels than cells without the virus. Transfection of full-length LANA, but not the C terminus alone, greatly induced RING3 gene expression, and LANA and RING3 co-localized even in the transfected cells, in the absence of KSHV/HHV-8 viral DNA. High levels of LANA expression led to the disappearance of heterochromatin in both human and mouse cells. We suggest that LANA and RING3 may create a local euchromatic microenvironment around the viral episomes that are anchored to the heterochromatin.

scholarly journals Epidemiology and Genetic Variability of HHV-8/KSHV among Rural Populations and Kaposi’s Sarcoma Patients in Gabon, Central Africa. Review of the Geographical Distribution of HHV-8 K1 Genotypes in Africa

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 175
Author(s):  
Antony Idam Mamimandjiami ◽  
Augustin Mouinga-Ondémé ◽  
Jill-Léa Ramassamy ◽  
Délia Doreen Djuicy ◽  
Philippe V. Afonso ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Molecular Diversity ◽  
Central Africa ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Buffy Coat ◽  
Rural Populations ◽  
Herpesvirus 8 ◽  
Nuclear Antigens ◽  
Polymerase Chain

Human herpesvirus 8 (HHV-8) is the etiological agent of all forms of Kaposi’s sarcoma (KS). K1 gene studies have identified five major molecular genotypes with geographical clustering. This study described the epidemiology of HHV-8 and its molecular diversity in Gabon among Bantu and Pygmy adult rural populations and KS patients. Plasma antibodies against latency-associated nuclear antigens (LANA) were searched by indirect immunofluorescence. Buffy coat DNA samples were subjected to polymerase chain reaction (PCR) to obtain a K1 gene fragment. We studied 1020 persons; 91% were Bantus and 9% Pygmies. HHV-8 seroprevalence was 48.3% and 36.5% at the 1:40 and 1:160 dilution thresholds, respectively, although the seroprevalence of HHV-8 is probably higher in Gabon. These seroprevalences did not differ by sex, age, ethnicity or province. The detection rate of HHV-8 K1 sequence was 2.6% by PCR. Most of the 31 HHV-8 strains belonged to the B genotype (24), while the remaining clustered within the A5 subgroup (6) and one belonged to the F genotype. Additionally, we reviewed the K1 molecular diversity of published HHV-8 strains in Africa. This study demonstrated a high seroprevalence of HHV-8 in rural adult populations in Gabon and the presence of genetically diverse strains with B, A and also F genotypes.

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