World Society for Virology first international conference: Tackling global virus epidemics.

This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16-18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered 140 on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.

Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes.

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical ß-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1.

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function.

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. "Bright and early" events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.

Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage.

DNA damage can be sensed as a danger-associated molecular pattern by the innate immune system. Here we find that keratinocytes and other human cells mount an innate immune response within hours of etoposide-induced DNA damage, which involves the DNA sensing adaptor STING but is independent of the cytosolic DNA receptor cGAS. This non-canonical activation of STING is mediated by the DNA binding protein IFI16, together with the DNA damage response factors ATM and PARP-1, resulting in the assembly of an alternative STING signaling complex that includes the tumor suppressor p53 and the E3 ubiquitin ligase TRAF6. TRAF6 catalyzes the formation of K63-linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. We propose that STING acts as a signaling hub that coordinates a transcriptional response depending on its mode of activation.

ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle-viral latency and the productive lytic cycle-and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, in which they escape immune detection by restricting viral gene expression. Importantly, however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B [ARID3B]) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle-dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.