Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Intrinsic p53 Activation Restricts Gammaherpesvirus-Driven Germinal Center B Cell Expansion during Latency Establishment.

Gammaherpesviruses (GHV) are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center (GC) B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of GHV pathogenesis, we demonstrate in vivo that tumor suppressor p53 is activated specifically in B cells that are latently infected by MHV68. In the absence of p53, the early expansion of MHV68 latency was greatly increased, especially in GC B cells, a cell-type whose proliferation was conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of GC B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that EBV-encoded latent membrane protein 1 (LMP1) similarly triggers a p53 response in primary B cells. Our data highlight a model in which GHV latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53. IMPORTANCE: Gammaherpesviruses cause lifelong infections of their hosts, commonly referred to as latency, that can lead to cancer. Latency establishment benefits from the functions of viral proteins that augment and amplify B cell activation, proliferation, and differentiation signals. In uninfected cells, off-schedule cellular differentiation would typically trigger anti-proliferative responses by effector proteins known as tumor suppressors. However, tumor suppressor responses to gammaherpesvirus manipulation of cellular processes remain understudied, especially those that occur during latency establishment in a living organism. Here we identify p53, a tumor suppressor commonly mutated in cancer, as a host factor that limits virus-driven B cell proliferation and differentiation, and thus, viral colonization of a host. We demonstrate that p53 activation occurs in response to viral latency proteins that induce B cell activation. This work informs a gap in our understanding of intrinsic cellular defense mechanisms that restrict lifelong GHV infection.

Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice.

Gammaherpesviruses (GHVs) are oncogenic viruses that establish lifelong infections and are significant causes of human morbidity and mortality. While several vaccine strategies to limit GHV infection and disease are in development, there are no FDA-approved vaccines for human GHVs. As a new approach to gammaherpesvirus vaccination, we developed and tested a replication-dead virus (RDV) platform, using murine gammaherpesvirus 68 (MHV68), a well-established mouse model for gammaherpesvirus pathogenesis studies and preclinical therapeutic evaluations. We employed codon-shuffling-based complementation to generate revertant-free RDV lacking expression of the essential replication and transactivator protein (RTA) encoded by ORF50 to arrest viral gene expression early after de novo infection. Inoculation with RDV-50.stop exposes the host to intact virion particles and leads to limited lytic gene expression in infected cells. Prime-boost vaccination of mice with RDV-50.stop elicited virus-specific neutralizing antibody and effector T cell responses in the lung and spleen. Vaccination with RDV-50.stop resulted in a near complete abolishment of virus replication in the lung 7 days post-challenge and virus reactivation from spleen 16 days post-challenge with WT MHV68. Ifnar1-/- mice, which lack the type I interferon receptor, exhibit severe disease upon infection with WT MHV68. RDV-50.stop vaccination of Ifnar1-/- mice prevented wasting and mortality upon challenge with WT MHV68. These results demonstrate that prime-boost vaccination with a GHV that is unable to undergo lytic replication offers protection against acute replication, reactivation, and severe disease upon WT virus challenge.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian Uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect, unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9 , and the viral DNA polymerase processivity factor, vPPF encoded by ORF59 . MHV68 vUNG colocalized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone, or in combination. Last, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE: Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo . In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus associated cancers.

A longitudinal study of SARS-CoV-2 antibody seroprevalence and mitigation behaviors among college students at an Arkansas University.

Objective: Assess university students' SARS-CoV-2 antibody seroprevalence and mitigation behaviors over time. Participants: Randomly selected college students (N = 344) in a predominantly rural Southern state. Methods: Participants provided blood samples and completed self-administered questionnaires at three timepoints over the academic year. Adjusted odds ratios and 95% confidence intervals were estimated from logistic regression analyses. Results: SARS-CoV-2 antibody seroprevalence was 18.2% in September 2020, 13.1% in December, and 45.5% in March 2021 (21% for those with no vaccination history). SARS-CoV-2 antibody seroprevalence was associated with large social gatherings, staying local during the summer break, symptoms of fatigue or rhinitis, Greek affiliation, attending Greek events, employment, and using social media as the primary COVID-19 information source. In March 2021, seroprevalence was associated with receiving at least one dose of a COVID-19 vaccination. Conclusion: SARS-CoV-2 seroprevalence was higher in this population of college students than previous studies. Results can assist leaders in making informed decisions as new variants threaten college campuses.

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

Lytic Replication and Reactivation from B Cells Is Not Required for Establishing or Maintaining Gammaherpesvirus Latency In Vivo.

Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced; however, the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Treatment of infected mice with lipopolysaccharide (LPS), which promotes B cell activation and MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir to prevent reactivation. Together, these data demonstrate that productive viral replication in B cells is not required for MHV68 latency establishment and support the hypothesis that B cell proliferation facilitates latency maintenance in vivo in the absence of reactivation. IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within B cells.

Temporal Variations in Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Infections by Race and Ethnicity in Arkansas.

Background: The aim of this study was to estimate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates in the small rural state of Arkansas, using SARS-CoV-2 antibody prevalence as an indicator of infection. Methods: We collected residual serum samples from adult outpatients seen at hospitals or clinics in Arkansas for non-coronavirus disease 2019 (COVID-19)-related reasons. A total of 5804 samples were identified over 3 time periods: 15 August-5 September 2020 (time period 1), 12 September-24 October 2020 (time period 2), and 7 November-19 December 2020 (time period 3). Results: The age-, sex-, race-, and ethnicity-standardized SARS-CoV-2 seroprevalence during each period, from 2.6% in time period 1 to 4.1% in time period 2 and 7.4% in time period 3. No statistically significant difference in seroprevalence was found based on age, sex, or residence (urban vs rural). However, we found higher seroprevalence rates in each time period for Hispanics (17.6%, 20.6%, and 23.4%, respectively) and non-Hispanic Blacks (4.8%, 5.4%, and 8.9%, respectively) relative to non-Hispanic Whites (1.1%, 2.6%, and 5.5%, respectively). Conclusions: Our data imply that the number of Arkansas residents infected with SARS-CoV-2 rose steadily from 2.6% in August to 7.4% in December 2020. There was no statistical difference in seroprevalence between rural and urban locales. Hispanics and Blacks had higher rates of SARS-CoV-2 antibodies than Whites, indicating that SARS-CoV-2 spread disproportionately in racial and ethnic minorities during the first year of the COVID-19 pandemic.

Oral Shedding of an Oncogenic Virus Alters the Oral Microbiome in HIV+ Patients.

Kaposi's Sarcoma (KS) caused by Kaposi's sarcoma-associated herpesvirus (KSHV) continues to be the most common AIDS-associated tumor. Involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. Numerous types of cancer are associated with the alterations of in components of the microbiome. However, little is known about how KSHV coinfection affects the oral microbiome in HIV+ patients, especially in a "pre-cancer" niche. Using 16S rRNA pyrosequencing, we found that oral shedding of KSHV correlated with altered oral microbiome signatures in HIV+ patients, including a reduction in the microbiota diversity, changing the relative composition of specific phyla and species, and regulating microbial functions. Furthermore, we found that Streptococcus sp., one of the most increased species in the oral cavity of HIV+/KSHV+ patients, induced KSHV lytic reactivation in primary oral cells. Together, these data indicate that oral shedding of KSHV may manipulate the oral microbiome to promote viral pathogenesis and tumorigenesis especially in immunocompromised patients.

State-wide random seroprevalence survey of SARS-CoV-2 past infection in a southern US State, 2020.

The purpose of this cross-sectional study was to estimate the proportion of Arkansas residents who were infected with the SARS-CoV-2 virus between May and December 2020 and to assess the determinants of infection. To estimate seroprevalence, a state-wide population-based random-digit dial sample of non-institutionalized adults in Arkansas was surveyed. Exposures were age, sex, race/ethnicity, education, occupation, contact with infected persons, comorbidities, height, and weight. The outcome was past COVID-19 infection measured by serum antibody test. We found a prevalence of 15.1% (95% CI: 11.1%, 20.2%) by December 2020. Seropositivity was significantly elevated among participants who were non-Hispanic Black, Hispanic (prevalence ratio [PRs]:1.4 [95% CI: 0.8, 2.4] and 2.3 [95% CI: 1.3, 4.0], respectively), worked in high-demand essential services (PR: 2.5 [95% CI: 1.5, 4.1]), did not have a college degree (PR: 1.6 [95% CI: 1.0, 2.4]), had an infected household or extra-household contact (PRs: 4.7 [95% CI: 2.1, 10.1] and 2.6 [95% CI: 1.2, 5.7], respectively), and were contacted in November or December (PR: 3.6 [95% CI: 1.9, 6.9]). Our results indicate that by December 2020, one out six persons in Arkansas had a past SARS-CoV-2 infection.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Development of ACE2 autoantibodies after SARS-CoV-2 infection.

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.

Deletion of Murine Gammaherpesvirus Gene M2 in Activation-Induced Cytidine Deaminase-Expressing B Cells Impairs Host Colonization and Viral Reactivation.

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

Identification of new antiviral agents against Kaposi's sarcoma-associated herpesvirus (KSHV) by high-throughput drug screening reveals the role of histamine-related signaling in promoting viral lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.

Expression of PD-1 and PD-Ls in Kaposi's sarcoma and regulation by oncogenic herpesvirus lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several cancers such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). PD-1/PD-Ls immune checkpoint molecules play important roles in cancer cell immune escape. The expression of PD-1/PD-Ls and their regulation by oncogenic viruses, in particular KSHV, remain largely undefined. Here we demonstrate strong PD-1/PD-L1/PD-L2 expression in KS tissues from a cohort of HIV + patients. We found that induction of KSHV lytic reactivation significantly upregulates PD-L1 expression on infected tumor cells, potentially through several major cellular signaling pathways and IL-1ß, which may represent a novel mechanism for virus-associated tumor cell immune escape.

IRF4 promotes Epstein-Barr virus activation in Burkitt's lymphoma cells.

Epstein-Barr virus (EBV) establishes a life-long latency in memory B cells, whereas plasma cell differentiation is linked to EBV lytic reactivation from latently infected B cells. EBV lytic replication is mediated by the two immediate-early switch proteins Zta and RTA. Both plasma cell transcription factors XBP-1 and Blimp-1 have been shown to enable the triggering of EBV lytic reactivation by activating the transcription of Zta or RTA. Here we show that interferon regulatory factor 4 (IRF4), another plasma cell transcription factor that is either not expressed or expressed at a low level in EBV-positive Burkitt's lymphoma (BL) cells, can activate the promoters of EBV Zta and RTA, but is not sufficient to elicit EBV lytic reactivation in latently infected BL cells. However, ectopic IRF4 expression can augment EBV lytic gene expression induced by anti-immunoglobulin (anti-Ig) or sodium butyrate treatment in all tested lymphoma cells, whereas IRF4 knockout in Raji cells, the only BL cell line with detectable endogenous IRF4 expression, abolishes EBV lytic gene expression induced by anti-Ig, and this is accompanied by the reduction of Blimp-1 expression, whose overexpression, in turn, can rescue EBV lytic gene expression in IRF4 knockout Raji cells. Furthermore, IRF4 knockout impairs B cell receptor (BCR) signalling activation, which is required for BCR-mediated EBV reactivation. Altogether, these results demonstrate that IRF4 facilitates EBV lytic reactivation in BL cells, which involves the regulation of Blimp-1 expression and BCR signalling pathways.

The TRiC chaperonin controls reovirus replication through outer-capsid folding.

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus-host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC; also called CCT for chaperonin containing TCP-1) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves folding the viral σ3 major outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related non-enveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.