scholarly journals Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Ken-ichi Nakajima ◽  
Sara Guevara-Plunkett ◽  
Frank Chuang ◽  
Kang-Hsin Wang ◽  
Yuanzhi Lyu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Infected Cell ◽  
Kaposi's Sarcoma ◽  
Individual Cell ◽  
Kaposi’S Sarcoma ◽  
Cell Populations ◽  
Replication Stage ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Molecular mechanisms of Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named “Rainbow-KSHV,” that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

scholarly journals Epstein-Barr Virus Latent Gene Expression in Primary Effusion Lymphomas Containing Kaposi's Sarcoma–Associated Herpesvirus/Human Herpesvirus-8

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1186-1191 ◽  
Author(s):  
Marcelo G. Horenstein ◽  
Roland G. Nador ◽  
Amy Chadburn ◽  
Elizabeth M. Hyjek ◽  
Giorgio Inghirami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Barr Virus ◽  
Low Levels ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Primary effusion (body cavity–based) lymphoma (PEL) is a recently recognized subtype of malignant lymphoma that exhibits distinctive clinical and biological features, most notably its usual infection with the Kaposi's sarcoma–associated herpesvirus (KSHV). The vast majority of cases also contain Epstein-Barr virus (EBV). This dual viral infection is the first example of a consistent dual herpesviral infection in a human neoplasm and provides a unique model to study viral interactions. We analyzed the pattern of EBV latent gene expression to determine the pathogenic role of this agent in PELs. We examined five PELs coinfected with EBV and KSHV by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. EBER1 mRNA, a consistent marker of viral latency, was positive in all PEL cases, although at lower levels than in the non-PEL controls due to EBER1 expression by only a variable subset of lymphoma cells. Qp-initiated mRNA, encoding only EBNA1 and characteristic of latencies I and II, was positive in all PEL cases. Wp- and Cp-initiated mRNAs, encoding all EBNAs and characteristic of latency III, were negative in all cases. LMP1 mRNA, expressed in latencies II and III, was present in three cases of PEL, although at very low levels that were not detectable at the protein level by immunohistochemistry. Low levels of LMP2A mRNA were detected in all cases. BZLF1, an early-intermediate lytic phase marker, was weakly positive in four cases, suggesting a productive viral infection in a very small proportion of cells, which was confirmed by ZEBRA antigen expression. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels.

scholarly journals The Interaction between ORF18 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-Associated Herpesvirus

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Angelica F. Castañeda ◽  
Britt A. Glaunsinger
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene ◽  
Central Component ◽  
Gene Promoters ◽  
Late Gene Expression ◽  
Late Genes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTIn the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral assembly and whose transcription initiates only after the onset of viral DNA replication. The vTAs in Kaposi’s sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins and how they coordinate to activate late gene promoters remain largely unknown. Here, we performed a comprehensive mutational analysis of the conserved residues in ORF18, which is a highly interconnected vTA component. Surprisingly, the mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical as even small disruptions in this complex result in profound defects in KSHV late gene expression.IMPORTANCEKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma and other B-cell cancers and remains a leading cause of death in immunocompromised individuals. A key step in the production of infectious virions is the transcription of viral late genes, which generates capsid and structural proteins and requires the coordination of six viral proteins that form a complex. The role of these proteins during transcription complex formation and the importance of protein-protein interactions are not well understood. Here, we focused on a central component of the complex, ORF18, and revealed that disruption of its interaction with even a single component of the complex (ORF30) prevents late gene expression and completion of the viral lifecycle. These findings underscore how individual interactions between the late gene transcription components are critical for both the stability and function of the complex.

scholarly journals The Kaposi’s Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Muzammel Haque ◽  
K. G. Kousoulas
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Proteasome Pathway ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTHypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi’s sarcoma (KS) and other AIDS-related malignancies. Kaposi’s sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCEHypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

scholarly journals Epstein-Barr Virus Latent Gene Expression in Primary Effusion Lymphomas Containing Kaposi's Sarcoma–Associated Herpesvirus/Human Herpesvirus-8

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1186-1191 ◽  
Author(s):  
Marcelo G. Horenstein ◽  
Roland G. Nador ◽  
Amy Chadburn ◽  
Elizabeth M. Hyjek ◽  
Giorgio Inghirami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Barr Virus ◽  
Low Levels ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Primary effusion (body cavity–based) lymphoma (PEL) is a recently recognized subtype of malignant lymphoma that exhibits distinctive clinical and biological features, most notably its usual infection with the Kaposi's sarcoma–associated herpesvirus (KSHV). The vast majority of cases also contain Epstein-Barr virus (EBV). This dual viral infection is the first example of a consistent dual herpesviral infection in a human neoplasm and provides a unique model to study viral interactions. We analyzed the pattern of EBV latent gene expression to determine the pathogenic role of this agent in PELs. We examined five PELs coinfected with EBV and KSHV by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. EBER1 mRNA, a consistent marker of viral latency, was positive in all PEL cases, although at lower levels than in the non-PEL controls due to EBER1 expression by only a variable subset of lymphoma cells. Qp-initiated mRNA, encoding only EBNA1 and characteristic of latencies I and II, was positive in all PEL cases. Wp- and Cp-initiated mRNAs, encoding all EBNAs and characteristic of latency III, were negative in all cases. LMP1 mRNA, expressed in latencies II and III, was present in three cases of PEL, although at very low levels that were not detectable at the protein level by immunohistochemistry. Low levels of LMP2A mRNA were detected in all cases. BZLF1, an early-intermediate lytic phase marker, was weakly positive in four cases, suggesting a productive viral infection in a very small proportion of cells, which was confirmed by ZEBRA antigen expression. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels.

scholarly journals A nuclear role for Kaposi's sarcoma-associated herpesvirus-encoded K13 protein in gene regulation

Oncogene ◽  
2008 ◽  
Vol 27 (39) ◽  
pp. 5243-5253 ◽  
Author(s):  
H Matta ◽  
V Punj ◽  
S Schamus ◽  
L Mazzacurati ◽  
A M Chen ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

scholarly journals Principal Role of TRAP/Mediator and SWI/SNF Complexes in Kaposi's Sarcoma-Associated Herpesvirus RTA-Mediated Lytic Reactivation

2003 ◽  
Vol 23 (6) ◽  
pp. 2055-2067 ◽  
Author(s):  
Yousang Gwack ◽  
Hwa Jin Baek ◽  
Hiroyuki Nakamura ◽  
Sun Hwa Lee ◽  
Michael Meisterernst ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Viral Gene Expression ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Viral Gene ◽  
Transcription Activator ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. The RTA transcription activator of Kaposi's sarcoma-associated herpesvirus (KSHV) acts as a molecular switch for lytic reactivation. Here we demonstrate that KSHV RTA recruits CBP, the SWI/SNF chromatin remodeling complex, and the TRAP/Mediator coactivator into viral promoters through interactions with a short acidic sequence in the carboxyl region and that this recruitment is essential for RTA-dependent viral gene expression. The Brg1 subunit of SWI/SNF and the TRAP230 subunit of TRAP/Mediator were shown to interact directly with RTA. Consequently, genetic ablation of these interactions abolished KSHV lytic replication. These results demonstrate that the recruitment of CBP, SWI/SNF, and TRAP/Mediator complexes by RTA is the principal mechanism to direct well-controlled viral gene expression and thereby viral lytic reactivation.

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