Virus–Host Interplay Between Poly (ADP-Ribose) Polymerase 1 and Oncogenic Gammaherpesviruses

The gammaherpesviruses, include the Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, and murine gammaherpesvirus 68. They establish latent infection in the B lymphocytes and are associated with various lymphoproliferative diseases and tumors. The poly (ADP-ribose) polymerase-1 (PARP1), also called ADP-ribosyltransferase diphtheria-toxin-like 1 (ARTD1) is a nuclear enzyme that catalyzes the transfer of the ADP-ribose moiety to its target proteins and participates in important cellular activities, such as the DNA-damage response, cell death, transcription, chromatin remodeling, and inflammation. In gammaherpesvirus infection, PARP1 acts as a key regulator of the virus life cycle: lytic replication and latency. These viruses also develop various strategies to regulate PARP1, facilitating their replication. This review summarizes the roles of PARP1 in the viral life cycle as well as the viral modulation of host PARP1 activity and discusses the implications. Understanding the interactions between the PARP1 and oncogenic gammaherpesviruses may lead to the identification of effective therapeutic targets for the associated diseases.

IKKalpha-Mediated Non-canonical NF-kappaB Signaling is Required to Support Murine Gammaherpesvirus 68 Latency In Vivo

Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68.

Age-associated B cells are critical for the control of latent gammaherpesvirus 68 infection

Age-associated B cells (ABCs; CD19+CD11c+T-bet+) are increased during an array of viral infections, though their role during viral latency is unexplored. Here, we use murine gammaherpesvirus 68 (γHV68), a homolog of Epstein-Barr virus that latently infects B cells, to demonstrate that ABCs are necessary for the effective control of gamma-herpesvirus latency. We observe that ABCs expand in the spleen during acute infection and persist at least 150 days post-infection. During acute and latent infection ABCs secrete IFNγ and TNF. Using a strain of γHV68 that is cleared following acute infection, we show that ABCs persist in the absence of latent virus, though they secrete less IFNγ and TNF. With a fluorescent virus we demonstrate that ABCs are infected with γHV68 at similar rates to other mature B cells. We find that mice without ABCs display defects in anti-viral IgG2a/c antibodies and are less able to maintain γHV68 latency when challenged with heterologous infection. Together, these results indicate that ABCs are a persistent subset during latent viral infection that controls γHV68 reactivation from latency.

Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68

Gammaherpesviruses are an important class of oncogenic pathogens that are exquisitely evolved to their respective hosts. As such, the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) do not naturally infect nonhuman primates or rodents. There is a clear need to fully explore mechanisms of gammaherpesvirus pathogenesis, host control, and immune evasion in the host. A gammaherpesvirus pathogen isolated from murid rodents was first reported in 1980; 40 years later, murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68) infection of laboratory mice is a well-established pathogenesis system recognized for its utility in applying state-of-the-art approaches to investigate virus-host interactions ranging from the whole host to the individual cell. Here, we highlight recent advancements in our understanding of the processes by which MHV68 colonizes the host and drives disease. Lessons that inform KSHV and EBV pathogenesis and provide future avenues for novel interventions against infection and virus-associated cancers are emphasized.

RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

Purification of Murine Gammaherpesvirus 68 With Use of Differential Centrifugation

The method for separation of viral particles in a concentrated form from the environment is called virus purification. Viruses are required to be purified for a range of studies in which it is necessary to distinguish the properties or structure of a virus from the host cells or culture media, including analysis of viral polypeptide structures and membrane glycoprotein function. Our objective was to purify murine gammaherpesvirus 68 (MHV-68, MuHV-4) using the centrifuge, equipment and other materials available in our laboratory. After infection of baby hamster kidney 21 (BHK-21) cells with MHV-68 with the multiplicity of infection (MI) of 0.01 and following virus multiplication, we repeatedly froze and thawed the cell culture to disrupt the cells and release the virus particles into the culture medium. We used low-speed centrifugation (3000 rpm at 4°C) to separate the viral particles from cell debris. Subsequently, we transferred the supernatant containing virus particles to a fresh centrifuge tube and centrifuged at a speed of 8000 rpm (8801 g) and 11,000 rpm (=16,639 g) and at 4°C. We tested different centrifugation durations of 2, 4, 6 and 8 hours. To evaluate the quality of the obtained purified MHV-68 virus by this method and compare it to purified MHV-68 sample acquired by conventional ultracentrifugation on sucrose cushion (30%, w/v), we used the SDS-PAGE separation method using a 4%–20% (w/v) and 6%–14% (w/v) gradient gel. We obtained the best results with 6-hour-long centrifugation at 11,000 rpm. In conclusion, we managed to optimise virus purification method using the equipment available in our laboratory and prepared purified MHV-68 virus in sufficient concentration for determination of MHV-68 virus proteins.

Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1–Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1–Nup98, the ORF10–Rae1–Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10–RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1–Nup98 in regulating host mRNA export.

KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA’s acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.

p53 Controls Murine Gammaherpesvirus Latency and Prevents Infection-Associated IgH/c-Myc Translocations

ABSTRACTGammaherpesviruses (GHVs) establish life-long infections and cause cancer in humans and other animals. To facilitate chronic infection, GHV oncoproteins promote cellular proliferation and differentiation. Aberrant cell-cycle progression driven by viral oncogenes should trigger activation of tumor suppressor p53, unless p53 is functionally deactivated during GHV latency establishment. However, interactions of GHVs with the p53 pathway during the establishment and maintenance of latent infection are poorly defined. Here we demonstrate in vivo that p53 is induced specifically in infected cells during latency establishment by murine gammaherpesvirus 68 (MHV68). In the absence of p53, MHV68 latency establishment was significantly increased, especially in germinal center B cells, and correlated with enhanced cellular proliferation. However, enhanced latency was not sustainable, and MHV68 exhibited a defect in long-term latency maintenance in p53-deficient mice. Moreover, IgH/c-Myc translocations were readily detected in B cells from infected p53-null mice indicating virus-driven genomic instability. These data demonstrate that p53 intrinsically restricts MHV68 latency establishment and reveal a paradigm in which a host restriction factor provides a long-term benefit to a chronic pathogen by limiting infection-associated damage.