Kaposi's Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny Viruses

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.

Section: Resultsmentioning

confidence: 99%

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

Nakajima

Guevara-Plunkett

Chuang

et al. 2020

“…By mass spectrometry analysis of mature KSHV virions, several tegument proteins, such as open reading frame 33 (ORF33), ORF38, ORF45, ORF52, ORF55, ORF63, ORF64, and ORF75, have been identified (11). Among these tegument proteins, some are important for innate immune modulation to facilitate the establishment of persistent infection (12)(13)(14)(15)(16)(17), and some are involved in virion tegumentation, assembly, and trafficking (18)(19)(20)(21)(22)(23). However, how these tegument proteins are packaged into virions is still largely unknown.…”

mentioning

confidence: 99%

Novel Role of vBcl2 in the Virion Assembly of Kaposi's Sarcoma-Associated Herpesvirus

Liang

Wei

Chung

et al. 2018

The viral Bcl-2 homolog (vBcl2) of Kaposi's sarcoma-associated herpesvirus (KSHV) displays efficient antiapoptotic and antiautophagic activity through its central BH3 domain, which functions to prolong the life span of virus-infected cells and ultimately enhances virus replication and latency. Independent of its antiapoptotic and antiautophagic activity, vBcl2 also plays an essential role in KSHV lytic replication through its amino-terminal amino acids (aa) 11 to 20. Here, we report a novel molecular mechanism of vBcl2-mediated regulation of KSHV lytic replication. vBcl2 specifically bound the tegument protein open reading frame 55 (ORF55) through its amino-terminal aa 11 to 20, allowing their association with virions. Consequently, the vBcl2 peptide derived from vBcl2 aa 11 to 20 effectively disrupted the interaction between vBcl2 and ORF55, inhibiting the incorporation of the ORF55 tegument protein into virions. This study provides new insight into vBcl2's function in KSHV virion assembly that is separable from its inhibitory role in host apoptosis and autophagy. KSHV, an important human pathogen accounting for a large percentage of virally caused cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish lifelong persistent infection. Upon viral infection, infected cells can go through programmed cell death, including apoptosis and autophagy, which plays an effective role in antiviral responses. To counter the host response, KSHV vBcl2 efficiently blocks apoptosis and autophagy to persist for the life span of virus-infected cells. Besides its anti-programmed-cell-death activity, vBcl2 also interacts with the ORF55 tegument protein for virion assembly in infected cells. Interestingly, the vBcl2 peptide disrupts the vBcl2-ORF55 interaction and effectively inhibits KSHV virion assembly. This study indicates that KSHV vBcl2 harbors at least three genetically separable functions to modulate both host cell death signaling and virion production and that the vBcl2 peptide can be developed as an anti-KSHV therapeutic application.

“…Nevertheless, we also found that ORF52 is able to induce alterations in the MT cytoskeleton not only in hTERT-RhF but also in 293T cells (not shown), suggesting that the effects are not cell type specific. Of note, a recent report that appeared as we prepared the present study showed that KSHV ORF52 also associates with MTs ( 56 ). Although, in our hands, the effect from the overexpression of the KSHV ortholog is significantly less pronounced than that with RRV ORF52, this congruence suggests a likely conservation of function among gammaherpesviruses.…”

Section: Discussionmentioning

confidence: 49%

A Conserved Leucine Zipper Motif in Gammaherpesvirus ORF52 Is Critical for Distinct Microtubule Rearrangements

Loftus

Verville

Kedes

2017

Productive viral infection often depends on the manipulation of the cytoskeleton. Herpesviruses, including rhesus monkey rhadinovirus (RRV) and its close homolog, the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8), exploit microtubule (MT)-based retrograde transport to deliver their genomes to the nucleus. Subsequently, during the lytic phase of the life cycle, the maturing viral particles undergo orchestrated translocation to specialized regions within the cytoplasm, leading to tegumentation, secondary envelopment, and then egress. As a result, we hypothesized that RRV might induce changes in the cytoskeleton at both early and late stages of infection. Using confocal imaging, we found that RRV infection led to the thickening and acetylation of MTs emanating from the MT-organizing center (MTOC) shortly after viral entry and more pronounced and diffuse MT reorganization during peak stages of lytic gene expression and virion production. We subsequently identified open reading frame 52 (ORF52), a multifunctional and abundant tegument protein, as being the only virally encoded component responsible for these cytoskeletal changes. Mutational and modeling analyses indicated that an evolutionarily conserved, truncated leucine zipper motif near the N terminus as well as a strictly conserved arginine residue toward the C terminus of ORF52 play critical roles in its ability to rearrange the architecture of the MT cytoskeleton. Taken together, our findings combined with data from previous studies describing diverse roles for ORF52 suggest that it likely binds to different cellular components, thereby allowing context-dependent modulation of function.IMPORTANCE A thorough understanding of the processes governing viral infection includes knowledge of how viruses manipulate their intracellular milieu, including the cytoskeleton. Altering the dynamics of actin or MT polymerization, for example, is a common strategy employed by viruses to ensure efficient entry, maturation, and egress as well as the avoidance of antiviral defenses through the sequestration of key cellular factors. We found that infection with RRV, a homolog of the human pathogen KSHV, led to perinuclear wrapping by acetylated MT bundles and identified ORF52 as the viral protein underlying these changes. Remarkably, incoming virions were able to supply sufficient ORF52 to induce MT thickening and acetylation near the MTOC, potentially aiding in the delivery viral genomes to the nucleus. Although the function of MT alterations during late stages of infection requires further study, ORF52 shares functional and structural similarities with alphaherpesvirus VP22, underscoring the evolutionary importance of MT cytoskeletal manipulations for this virus family.