The gammaherpesvirus 68 viral cyclin facilitates expression of LANA

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.

The gammaherpesvirus 68 viral cyclin facilitates reactivation by promoting latent gene expression

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into more active lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on latent gene expression, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of latent gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential latent gene expression, and that a primary function of the viral cyclin is to promote latent gene expression within infected cells in vivo.AUTHOR SUMMARYGammaherpesviruses are ubiquitous viruses with oncogenic potential that establish latency for the life of the host. These viruses can emerge from latency through reactivation, a process that is controlled by the immune system. Control of viral latency and reactivation is thought to be critical to prevent γHV-associated disease. This study focuses on a virally-encoded cyclin that is required for reactivation from latency. By characterizing how the viral cyclin influences latent infection in pure cell populations, we find that the viral cyclin has a vital role in promoting viral gene expression during latency. This work provides new insight into the function of a virally encoded cyclin in promoting reactivation from latency.

MODERN ETHIOTROPIC CHEMOTHERAPY OF HUMAN CYTOMEGALOVIRUS INFECTION: CLINICAL EFFECTIVENESS, MOLECULAR MECHANISM OF ACTION, DRUG RESISTANCE, NEW TRENDS AND PROSPECTS. PART 2

A number of synthetic compounds, such as the nucleoside analog ganciclovir, its L-valine ester (a metabolic precursor of ganciclovir) and pyrophosphate analog foscarnet, are permitted for the treatment of HCMV-related diseases in the WHO European Region. The viral DNA- polymerase is used by all these drugs as a bio-target. However, the usage of standard anti-CMV therapy is accompanied by severe side effects, as well as the development of drug resistance in the virus, mainly in conditions of immunodeficiency. In this review, we focused on viral proteins of interest as new potential targets and their inhibitors, such as the inhibitor of human CMV terminology, lethermovir, which showed great activity in the third phase of clinical trials, inhibitors of viral cyclin-dependent kinase (maribavir, cyclopropavir ) and a number of compounds exhibiting anti-HCMV-activity, undergoing only preclinical trials in the experiment. Inclusion of new anti-CMV agents that are active against GСV/PFA/CDV-resistant strains of CMV into standard prophylactic and therapeutic regimens, will allow to increase the effectiveness of anti-CMV therapy, including in cases when standard therapy is ineffective. Areas covered: the international databases such as A MEDLINE, PubMed, eLIBRARY.RU, ClinicalTrials.gov., etc. with the purpose of obtaining information on compounds showing selective action against the human cytomegalovirus, the most promising for the development of drugs.

Host Tumor Suppressor p18INK4cFunctions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis

ABSTRACTGammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c(p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4cgene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCEGammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.

A Conserved Gammaherpesvirus Cyclin Specifically Bypasses Host p18INK4cTo Promote Reactivation from Latency

ABSTRACTGammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitorsin vitro; however, thein vivosignificance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27Kip1and p18INK4c, two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27Kip1and p18INK4c. Further analysis revealed that genetic ablation of only p18INK4calleviated the requirement for the viral cyclin for reactivation from latency. p18INK4cregulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18INK4cheterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18INK4cfunction in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18INK4c, revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor.IMPORTANCEThe gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins.