Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation.

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

Reactivation of Epstein-Barr virus among intensive care patients: a prospective observational study.

PURPOSE: Herpesvirus reactivation has been documented among patients in the intensive care unit (ICU) and is associated with increased morbidity and mortality, particularly for cytomegalovirus (CMV). Epstein-Barr virus (EBV) has been poorly studied despite >95% of the population being seropositive. Our preliminary study suggested an association between EBV reactivation and increased morbidity and mortality. This study aimed to investigate this association among patients admitted to the ICU. METHODS: In this multicenter prospective study, polymerase chain reaction was performed to quantify EBV in patients upon ICU admission and then twice a week during their stay. Follow-up was 90 days. RESULTS: The study included 129 patients; 70 (54.3%) had EBV reactivation. On day 90, there was no difference in mortality rates between patients with and without reactivation (25.7% vs 15.3%, p = 0.22). Patients with EBV reactivation at admission had increased mortality compared with those without reactivation and those with later reactivation. EBV reactivation was associated with increased morbidity. Patients with EBV reactivation had fewer ventilator-free days at day 28 than those without reactivation (18 [1-22] vs. 21 days [5-26], p = 0.037) and a higher incidence of acute respiratory distress syndrome (34.3% vs. 17%, p = 0.04), infections (92.9% vs. 78%, p = 0.03), and septic shock (58.6% vs. 32.2%, p = 0.004). More patients with EBV reactivation required renal replacement therapy (30% vs. 11.9%, p = 0.02). EBV reactivation was also associated with a more inflammatory immune profile. CONCLUSION: While EBV reactivation was not associated with increased 90-day mortality, it was associated with significantly increased morbidity.

Awakening the sleeping giant: Epstein-Barr virus reactivation by biological agents.

Epstein-Barr virus (EBV) may cause harm in immunocompromised conditions or on stress stimuli. Various chemical agents have been utilized to induce the lytic cycle in EBV-infected cells. However, apart from chemical agents and external stress stimuli, certain infectious agents may reactivate the EBV. In addition, the acute infection of other pathogens may provide suitable conditions for EBV to thrive more and planting the roots for EBV-associated pathologies. Various bacteria such as periodontal pathogens like Aggregatibacter, Helicobacter pylori, etc. have shown to induce EBV reactivation either by triggering host cells directly or indirectly. Viruses such as Human simplex virus-1 (HSV) induce EBV reactivation by HSV US3 kinase while other viruses such as HIV, hepatitis virus, and even novel SARS-CoV-2 have also been reported to cause EBV reactivation. The eukaryotic pathogens such as Plasmodium falciparum and Aspergillus flavus can also reactivate EBV either by surface protein interaction or as an impact of aflatoxin, respectively. To highlight the underexplored niche of EBV reactivation by biological agents, we have comprehensively presented the related information in this review. This may help to shedding the light on the research gaps as well as to unveil yet unexplored mechanisms of EBV reactivation.

[Guillain-Barré syndrome associated with Epstein-Barr virus and cytomegalovirus reactivation during treatment for chronic graft-versus-host disease after allogeneic bone marrow transplantation].

Guillain-Barré syndrome (GBS) is a rare neurological complication of allogeneic hematopoietic stem cell transplantation (HSCT). The pathogenesis of post-HSCT GBS is unclear. Here, we report a case of GBS coincident with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivation that occurred after HSCT in a patient with myelodysplastic syndrome. A 61-year-old man was admitted to our hospital because of gait disturbance due to lower limb muscle weakness, which arose during treatment for chronic graft-versus-host disease (GVHD) five months after allogeneic HSCT. He was diagnosed with GBS based on his clinical course, cerebrospinal fluid analysis, and a nerve conduction study. At that time, he exhibited EBV and CMV reactivation. GBS improved after intravenous injection of immunoglobulins. Our case suggests that reactivation of EBV and CMV during treatment for chronic GVHD may induce GBS, and that rapidly progressive muscular weakness coincident with EBV or CMV reactivation can be a diagnostic sign of GBS after allogeneic HSCT.

EBV Reactivation from Latency Is a Degrading Experience for the Host.

During reactivation from latency, gammaherpesviruses radically restructure their host cell to produce virion particles. To achieve this and thwart cellular defenses, they induce rapid degradation of cytoplasmic mRNAs, suppressing host gene expression. In this article, we review mechanisms of shutoff by Epstein-Barr virus (EBV) and other gammaherpesviruses. In EBV, canonical host shutoff is accomplished through the action of the versatile BGLF5 nuclease expressed during lytic reactivation. We explore how BGLF5 induces mRNA degradation, the mechanisms by which specificity is achieved, and the consequences for host gene expression. We also consider non-canonical mechanisms of EBV-induced host shutoff. Finally, we summarize the limitations and barriers to accurate measurements of the EBV host shutoff phenomenon.

[Reactivation of latent viruses in Neurology].

After establishing latent infection, some viruses can be reactivated by the alteration of host immunological conditions. First, we reviewed viruses that can cause neuronal damage by reactivation. Then we focused on the herpes simplex virus (HSV). The reactivation leads to neuronal damages through two possible mechanisms; "reactivation of a latent herpes virus" by which viruses can cause direct virus neurotoxicity, and "post-infectious immune inflammatory response" by which a focal reactivation of HSV leads to an inflammatory reaction. The former is radiologically characterized by cortical lesions, the latter is characterized by subcortical white matter lesions. We experienced a female, who underwent the right posterior quadrantectomy and then developed recurrent herpes encephalitis caused by herpes simplex reactivation, which pathologically demonstrated inflammation in the white matter, suggesting a post-infectious immune inflammatory response. The patient was successfully treated with immunosuppressants. The reactivation of the HSV is extremely rare in Japan. Neurologists should recognize this condition because this disorder will increase as epilepsy surgery gains more popularity.

Features of Epstein-Barr Virus and Cytomegalovirus Reactivation in Acute Leukemia Patients After Haplo-HCT With Myeloablative ATG-Containing Conditioning Regimen.

Background: Haploidentical donor hematopoietic cell transplantation (haplo-HCT) has become a preferred option for patients without HLA-matched donors, but it increases the risk of viral reactivations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are common viruses post-HCT, but limited data have been reported in the setting of haplo-HCT. Methods: We conducted a retrospective study enrolling acute leukemia patients who received haplo-HCT with myeloablative conditioning regimen employing ATG in our center from July 2014 to July 2017. All the patients enrolled were EBV-IgM and EBV-DNA negative but EBV-IgG positive, and so were their donors. The same went for CMV as well. Results: In total, 602 patients were recruited consisting of 331 with acute myeloid leukemia (AML) and 271 with acute lymphoblastic leukemia (ALL). One-year cumulative incidences of EBV (22.9% ± 2.4% vs. 27.4% ± 2.8%, P = 0.169) and CMV (24.7% ± 2.4% vs. 29.4% ± 2.8%, P = 0.190) reactivation were comparable between AML and ALL. EBV and CMV were independent risk factors for each other. In the AML group, male recipients [HR = 1.275, 95% CI (1.001-1.624), P = 0.049] and acute graft-versus-host disease [HR = 1.592, 95% CI (1.001-2.533), P = 0.049] were independent risk factors for EBV reactivation and CMV reactivation, respectively. CMV rather than EBV reactivation was related to a trend of worsened treatment-related mortality (TRM) (15.6% ± 0.1% vs. 10.2% ± 0.0%, P = 0.067) and progression-free survival (PFS) (60.6% ± 4.1% vs. 70.3% ± 2.3%, P = 0.073), while significant impacts were revealed only in the subgroup analysis. CMV reactivation resulted in a remarkable inferior 2-year overall survival (OS) (64.2% ± 5.7% vs. 77.6% ± 3.2%, P = 0.038) and PFS (55.0% ± 5.9% vs. 71.9% ± 3.4%, P = 0.042) in ALL patients. On the other hand, in the EBV+/CMV- subgroup, relapse was lower in ALL patients (8.2% ± 0.2% vs. 32.4% ± 0.8%, P = 0.010) compared with AML patients, which led to a superior 2-year OS (82.0% ± 6.2% vs. 60.3% ± 8.8%, P = 0.016) and PFS (74.5% ± 7.0% vs. 57.5% ± 8.4%, P = 0.036). Conclusion: We concluded that EBV and CMV reactivations were frequent in acute leukemia patients after haplo-HCT, with possibly distinctive risk factors from HLA-matched HCT. There could be a potential interaction between EBV and CMV, but impacts on transplant outcomes remained complex.

Impact of Cultured Neuron Models on α-Herpesvirus Latency Research.

A signature trait of neurotropic α-herpesviruses (α-HV) is their ability to establish stable non-productive infections of peripheral neurons termed latency. This specialized gene expression program is the foundation of an evolutionarily successful strategy to ensure lifelong persistence in the host. Various physiological stresses can induce reactivation in a subset of latently-infected neurons allowing a new cycle of viral productive cycle gene expression and synthesis of infectious virus. Recurring reactivation events ensure transmission of the virus to new hosts and contributes to pathogenesis. Efforts to define the molecular basis of α-HV latency and reactivation have been notoriously difficult because the neurons harboring latent virus in humans and in experimentally infected live-animal models, are rare and largely inaccessible to study. Increasingly, researchers are turning to cultured neuron infection models as simpler experimental platforms from which to explore latency and reactivation at the molecular level. In this review, I reflect on the strengths and weaknesses of existing neuronal models and briefly summarize the important mechanistic insights these models have provided. I also discuss areas where prioritization will help to ensure continued progress and integration.

Reactivation of EBV and CMV in Severe COVID-19-Epiphenomena or Trigger of Hyperinflammation in Need of Treatment? A Large Case Series of Critically ill Patients.

BACKGROUND: Reactivation of viruses such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are common in critically ill patients and have been described in patients with severe COVID-19. However, it is unclear whether these reactivations are associated with increased mortality and whether targeted treatments are beneficial. METHODS: In a retrospective single-center cohort study, patients with severe COVID-19 treated on our intensive care unit (ICU) were screened for EBV and CMV reactivation as detected by polymerase chain reaction. If present, patient characteristics, temporal connections to severe acute respiratory syndrome coronavirus 2 diagnosis and corticosteroid use, the use of targeted treatments as well as the course of disease and outcome were analyzed. As control group, non-COVID-19 patients with sepsis, treated within the same time period on our ICU, served as control group to compare incidences of viral reactivation. RESULTS: In 19 (16%) of 117 patients with severe COVID-19 treated on our ICU EBV reactivations were identified, comparable 18 (14%) of 126 in the non-COVID-19 control group (P = .672). Similarly, in 11 (9%) of 117 patients CMV reactivations were identified, comparable to the 16 (13%) of 126 in the non-COVID-19 sepsis patients (P = .296). The majority of EBV (58%) and CMV reactivations (55%) were detected in patients under systemic corticosteroid treatment. 7 (37%) of 19 patients with EBV reactivation survived the ICU stay, 2 (29%) of 7 patients with rituximab treatment and 5 (42%) of 12 patients without treatment (P = .568). Five (50%) of 10 patients with CMV reactivation survived the ICU stay, 5 (83%) of 6 patients with ganciclovir treatment and 0 of 4 patients without treatment (P = .048). Follow-up analysis in these patients showed that the initiation of treatment lead to decrease in viral load. CONCLUSION: Critically ill patients with COVID-19 are at a high risk for EBV and CMV reactivations. Whether these reactivations are a cause of hyperinflammation and require targeted treatment remains uncertain. However, in patients with clinical deterioration or signs of hyperinflammation targeted treatment might be beneficial and warrants further studying.

Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination.

Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.

What Is the Risk of Reactivation in Patients with Resolved and Past HBV Infection During Immunosuppressive Therapy If HBV-DNA Negative before Treatment?

BACKGROUND: Reactivation of Hepatitis B (HBVr) related to immunosuppressive drug therapy (ISDT) in patients with resolved and past infection is a challenging entity. The number of prospective long-term studies is limited. METHODS: Two groups of patients with resolved and past HBV infection were analyzed prospectively. The patients were further categorized as 266 patients receiving ISDT (group 1) and 246 patients receiving antineoplastic therapy (group 2). RESULTS: We did not detect any cases of HBVr among 108 patients receiving rituximab (71 of which were anti-HBc positive only), 111 patients receiving tumor necrosis factor inhibitors (66 of which were anti-HBc positive only), and 42 patients receiving high-dose glucocorticoids for more than 4 weeks (24 of which were anti-HBc positive only) during a mean follow-up time of more than 24 months. Subgroup analysis of the anti-HBs (+) patients showed that in group A (anti-HBs >1000 mIU/mL) the antibody levels did not change; in group B (anti-HBs between 100 and 1000 mIU/mL) the antibody levels changed non-significantly (P = .25), and in Group C (anti-HBs between 0 and 100 mIU/mL) the antibody levels declined significantly (P = .002). Furthermore, 16 patients in Group C had an anti-HBs loss during follow-up, but no HBVr was detected. CONCLUSION: The risk of HBVr by immunosuppressive therapy in this group may be lower than that suspected in the literature and anti- HBs levels may not seem to correlate with the risk of reactivation.

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation.

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman's Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y481EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y481EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y481EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.

Bone Marrow-Derived SH-SY5Y Neuroblastoma Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus Display Unique Infection Phenotypes and Growth Properties.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an important oncogenic virus previously shown to be neurotropic, but studies on neuronal cell infection and pathogenesis are still very limited. Here, we characterized the effects of KSHV infection on neuronal SH-SY5Y cells by the recombinant virus rKSHV.219, which expresses both green fluorescent protein (GFP) and red fluorescent protein (RFP) to reflect the latent and lytic phases of infection. We demonstrated that infected cells have a higher growth rate and that KSHV infection can be sustained. Interestingly, the infected cells can transition spontaneously back and forth between lytic and latent phases of infection, producing progeny viruses but without any adverse effects on cell growth. In addition, transcriptome analysis of viral and cellular genes in latent and lytic cells showed that unlike other infected cell lines, the latently infected cells expressed both latent and most, but not all, of the lytic genes required for infectious virion production. The viral genes uniquely expressed by the lytic cells were mainly involved in the early steps of virus binding. Some of the cellular genes that were deregulated in both latently and lytically infected cells are involved in cell adhesion, cell signal pathways, and tumorigenesis. The downregulated cellular CCDN1, PAX5, and NFASC and upregulated CTGF, BMP4, YAP1, LEF1, and HLA-DRB1 genes were found to be associated with cell adhesion molecules (CAMs), hippo signaling, and cancer. These deregulated genes may be involved in creating an environment that is unique in neuronal cells to sustain cell growth upon KSHV infection and not observed in other infected cell types. IMPORTANCE Our study has provided evidence that neuronal SH-SY5Y cells displayed unique cellular responses upon KSHV infection. Unlike other infected cells, this neuronal cell line displayed a higher growth rate upon infection and can spontaneously transition back and forth between latent and lytic phases of infection. Unlike other latently infected cells, a number of lytic genes were also expressed in the latent phase of infection in addition to the established latent viral genes. They may play a role in deregulating a number of host genes that are involved in cell signaling and tumorigenesis in order to sustain the infection and growth advantages for the cells. Our study has provided novel insights into KSHV infection of neuronal cells and a potential new model for further studies to explore the underlying mechanism in viral and host interactions for neuronal cells and the association of KSHV with neuronal diseases.

Human Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation.

Of the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.

Endoplasmic Reticulum-Shaping Atlastin Proteins Facilitate KSHV Replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) has two life cycle modes: the latent and lytic phases. The endoplasmic reticulum (ER) is the site for KSHV production. Furthermore, ER stress can trigger reactivation of KSHV. Little is known about the nature of the ER factors that regulate KSHV replication. Atlastin proteins (ATLs which include ATL1, ATL2, and ATL3) are large dynamin-related GTPases that control the structure and the dynamics of the ER membrane. Here, we show that ATLs can regulate KSHV lytic activation and infection. Overexpression of ATLs enhances KSHV lytic activation, whereas ATLs silence inhibits it. Intriguingly, we find that silencing of ATLs impairs the response of cells to ER stress, and ER stress can promote the lytic activation of KSHV. Our study establishes that ATLs plays a critically regulatory role in KSHV infection, thus expanding the known scope of biological processes controlled by ATLs to include KSHV infection.

Cervical cytomegalovirus reactivation, cytokines and spontaneous preterm birth in Kenyan women.

Genital cytomegalovirus (CMV) reactivation is common during the third trimester of pregnancy. We hypothesized that cervical CMV shedding may increase risk of spontaneous preterm birth (sPTB) through the release of inflammatory cytokines in the cervix. We conducted a nested case-control analysis to determine the relationship between CMV shedding and sPTB using data and samples from a prospective cohort study in western Kenya. Women who delivered between 28 + 0 and 33 + 6 weeks gestation were matched by gestational age at sample collection to controls who delivered ≥ 37 + 0 weeks. Levels of CMV DNA and interleukin (IL)-1 beta (ß), IL-6, IL-8 and tumor necrosis factor (TNF)-α were measured in cervical swabs. We used conditional logistic regression to assess relationships between CMV shedding, cervical cytokine levels and sPTB. Among 86 cases and 86 matched controls, cervical CMV levels were not significantly associated with sPTB [odds ratio (OR) = 1·23, 95% confidence interval (CI) = 0·59-2·56], but were significantly associated with higher levels of cervical IL-6 (ß = 0·15, 95% CI = 0·02-0·29) and TNF-α (ß = 0·14, 95% CI = 0·01-0·27). In univariate analysis, higher odds of sPTB was associated with higher cervical IL-6 levels (OR = 1·54, 95% CI = 1·00-2·38), but not with other cervical cytokines. In this cohort of Kenyan women, we did not find a significant association between cervical CMV shedding and sPTB before 34 weeks.

A Protocol to Engineer Bacteriophages for Live-Cell Imaging of Bacterial Prophage Induction Inside Mammalian Cells.

The gut microbiome is dominated by lysogens, bacteria that carry bacterial viruses (phages). Uncovering the function of phages in the microbiome and observing interactions between phages, bacteria, and mammalian cells in real time in specific cell types are limited by the difficulty of engineering fluorescent markers into large, lysogenic phage genomes. Here, we present a method to multiplex the engineering of life-cycle reporters into lysogenic phages and how to infect macrophages with engineered lysogens to study these interactions at the single-cell level. For complete details on the use and execution of this protocol, please refer to Bodner et al. (2020).

RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression.

Herpes simplex virus 1 (HSV-1) and HSV-2 can efficiently establish lifelong, transcriptionally silent latency states in sensory neurons to escape host detection. While host factors have previously been associated with long-range insulators in the viral genome, it is still unknown whether host transcription factors can repress viral genes more proximately to promote latency in dorsal root ganglion (DRG) neurons. Here, we assessed whether RUNX (runt-related transcription factor) transcription factors, which are critical in the development of sensory neurons, could be binding HSV-1 genome directly to suppress viral gene expression and lytic infection. Using previously published transcriptome sequencing data, we confirmed that mouse DRG neurons highly express Runx1 mRNA. Through computational analysis of HSV-1 and HSV-2 genomes, we observed that putative RUNX consensus binding sites (CBSs) were more enriched and more closely located to viral gene transcription start sites than would be expected by chance. We further found that RUNX CBSs were significantly more enriched among genomes of herpesviruses compared to those of nonherpesviruses. Utilizing an in vitro model of HSV-1 infection, we found that overexpressed RUNX1 could bind putative binding sites in the HSV-1 genome, repress numerous viral genes spanning all three kinetic classes, and suppress productive infection. In contrast, knockdown of RUNX1 in neuroblastoma cells induced viral gene expression and increased HSV-1 infection in vitro In sum, these data support a novel role for RUNX1 in directly binding herpesvirus genome, silencing the transcription of numerous viral genes, and ultimately limiting overall infection.IMPORTANCE Infecting 90% of the global population, HSV-1 and HSV-2 represent some of the most prevalent viruses in the world. Much of their success can be attributed to their ability to establish lifelong latent infections in the dorsal root ganglia (DRG). It is still largely unknown, however, how host transcription factors are involved in establishing this latency. Here, we report that RUNX1, expressed highly in DRG, binds HSV-1 genome, represses transcription of numerous viral genes, and suppresses productive in vitro infection. Our computational work further suggests this strategy may be used by other herpesviruses to reinforce latency in a cell-specific manner.

Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation.

Gammaherpesviruses have evolved various strategies to take advantage of host cellular factors or signaling pathways to establish a lifelong latent infection. Like the human gammaherpesvirus Epstein-Barr virus, murine gammaherpesvirus 68 (MHV68) establishes and maintains latency in the memory B cells during infection of laboratory mice. We have previously shown that MHV68 can immortalize fetal liver-derived B cells that induce lymphomas when injected into immunodeficient mice. Here we identify interleukin 16 (IL16) as a most abundantly expressed cytokine in MHV68-immortalized B cells and show that MHV68 infection elevates IL16 expression. IL16 is not important for MHV68 lytic infection but plays a critical role in MHV68 reactivation from latency. IL16 deficiency increases MHV68 lytic gene expression in MHV68-immortalized B cells and enhances reactivation from splenic latency. Correlatively, IL16 deficiency increases the frequency of MHV68-infected plasma cells that can be attributed to enhanced MHV68 reactivation. Furthermore, similar to TPA-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus, IL16 deficiency markedly induces Tyr705 STAT3 de-phosphorylation and elevates p21 expression, which can be counteracted by the tyrosine phosphatase inhibitor orthovanadate. Importantly, orthovanadate strongly blocks MHV68 lytic gene expression mediated by IL16 deficiency. These data demonstrate that virus-induced IL16 does not directly participate in MHV68 lytic replication, but rather inhibits virus reactivation to facilitate latent infection, in part through the STAT3-p21 axis.