Advances in Model Systems for Human Cytomegalovirus Latency and Reactivation

Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells.

RTA and LANA Competitively Regulate let-7a/RBPJ Signal to Control KSHV Replication

The recombination signal binding protein for immunoglobulin kappa J region (RBPJ) has a dual effect on Kaposi’s sarcoma-associated herpesvirus (KSHV) replication. RBPJ interaction with replication and transcription activator (RTA) is essential for lytic replication, while the interaction with latency-associated nuclear antigen (LANA) facilitates latent infection. Furthermore, our previous study found that LANA decreased RBPJ through upregulating miRNA let-7a. However, it is unclear whether RTA regulates the expression of RBPJ. Here, we show RTA increases RBPJ by decreasing let-7a. During KSHV replication, the RBPJ expression level was positively correlated with the RTA expression level and negatively correlated with the LANA expression level. The let-7a expression level was inverse to RBPJ. Knockdown of RBPJ inhibited the self-activation of RTA promoter and LANA promoter and weakened LANA’s inhibition of RTA promoter. Collectively, these findings indicate that RTA and LANA compete for let-7a/RBPJ signal to control the KSHV replication. Regulating the RBPJ expression level by RTA and LANA plays an important role during KSHV replication.

Mass Spectrometry-Based Identification of New Serum Biomarkers in Patients with Latent Infection Pulmonary Tuberculosis

Background: To screen specific metabolic markers serum metabolic biomarkers which can achieve the main monitoring indicators to evaluate the development from latent infection to active tuberculosis infection, and analysis its underlying mechanisms and functions. Methods: Four groups of serum, including healthy control, latent infection, drug sensitivity (DS), and drug resistant tuberculosis, were collected. The metabolites in all serum samples were extracted by oscillatory, deproteinization, and then were detected by LC-MS/MS analysis. Normalization by Pareto-scaling method, the difference analysis was carried out by Metaboanalyst 4.0 software, one-way ANOVA analysis among groups showed that p-value ≤0.05 was regarded as a different metabolite. To clarify the dynamic changes and functions of differential metabolites with disease progression, and explore its significance and mechanism as a marker by further cluster analysis, functional enrichment analysis, and relative content change analysis of differential metabolites. Results: There were 565 significantly different metabolites in four groups. Differential metabolites, including Indole-3-acetaldehyde, Theophylline, Inosine and Prostaglandin H2, etc., may be the key serum biomarkers to diagnose the period of latent infection of Mycobacterium tuberculosis (M. tuberculosis). which was closely related to Amino acid metabolism, Biosynthesis of other secondary metabolites, Nucleotide metabolism, Endocrine system, Immune system, Lipid metabolism, and Nervous system. Conclusion: Indole-3-acetaldehyde, Theophylline, Inosine, and Prostaglandin H2, the 4 metabolites may be potential markers diagnosing the period of latent infection of M. tuberculosis. Meanwhile, Inosine and Prostaglandin E1 can become potential biomarkers for the diagnosis of latent infection, and Theophylline and Cotinine 1 can be used as potential markers to monitor disease progression, which established strategy provided promising clinical application prospects for the development of disease assessment by combining small molecule metabolic markers to improve the sensitivity and specificity of disease diagnosis.

Targeting Non-Replicating Mycobacterium tuberculosis and Latent Infection: Alternatives and Perspectives (Mini-Review)

Latent tuberculosis infection (LTBI) represents a major challenge to curing TB disease. Current guidelines for LTBI management include only three older drugs and their combinations—isoniazid and rifamycins (rifampicin and rifapentine). These available control strategies have little impact on latent TB elimination, and new specific therapeutics are urgently needed. In the present mini-review, we highlight some of the alternatives that may potentially be included in LTBI treatment recommendations and a list of early-stage prospective small molecules that act on drug targets specific for Mycobacterium tuberculosis latency.

Antibody Generation and Immunogenicity Analysis of EBV gp42 N-Terminal Region

Epstein–Barr virus (EBV) is the first reported oncogenic virus and infects more than 90% of adults worldwide. EBV can establish a latent infection in B lymphocytes which is essential for persistence and transmission. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into a B cell. The N-terminal region of gp42 plays a key role in binding to gH/gL and triggering subsequent membrane fusion. However, no antibody has been reported to recognize this region and the immunogenicity of gp42 N-domain remains unknown. In the present study, we have generated a panel of nine mAbs against the gp42 N-terminal region (six mAbs to gp42-44-61aa and three mAbs to gp42-67-81aa). These mAbs show excellent binding activity and recognize different key residues locating on the gp42 N-domain. Among the nine mAbs, 4H7, 4H8 and 11G10 cross-react with rhLCV-gp42 while other mAbs specifically recognize EBV-gp42. Our newly obtained mAbs provide a useful tool for investigating the gp42 function and viral infection mechanism of γ-Herpesvirus. Furthermore, we assess the immunogenicity of the gp42 N-terminal region using the HBc149 particle as a carrier protein. The chimeric VLPs can induce high antibody titers and elicit neutralizing humoral responses to block EBV infection. More rational and effective designs are required to promote the gp42-N terminal region to become an epitope-based vaccine.

Latently KSHV-Infected Cells Promote Further Establishment of Latency upon Superinfection with KSHV

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related virus which engages in two forms of infection: latent and lytic. Latent infection allows the virus to establish long-term persistent infection, whereas the lytic cycle is needed for the maintenance of the viral reservoir and for virus spread. By using recombinant KSHV viruses encoding mNeonGreen and mCherry fluorescent proteins, we show that various cell types that are latently-infected with KSHV can be superinfected, and that the new incoming viruses establish latent infection. Moreover, we show that latency establishment is enhanced in superinfected cells compared to primary infected ones. Further analysis revealed that cells that ectopically express the major latency protein of KSHV, LANA-1, prior to and during infection exhibit enhanced establishment of latency, but not cells expressing LANA-1 fragments. This observation supports the notion that the expression level of LANA-1 following infection determines the efficiency of latency establishment and avoids loss of viral genomes. These findings imply that a host can be infected with more than a single viral genome and that superinfection may support the maintenance of long-term latency.

iTRAQ-based proteomics analysis of HCMV latency and reactivation in T98G cells

Human cytomegalovirus (HCMV) establishes a persistent/latent infection after primary infection, and host factor(s) plays a key role in regulating HCMV infection status. The spread of reactivated HCMV via the hematogenous or neural route usually results in severe diseases in newborns and immunocompromised individuals. As the primary reservoirs in vivo , cells of myeloid lineage have been utilized extensively to study HCMV infection. However, the molecular mechanism of HCMV latency/reactivation in neural cells is still poorly understood. We previously showed that HCMV infected T98G cells maintain a large number of viral genomes and support HCMV reactivation from latency upon cAMP/IBMX treatment. Here we employed iTRAQ-based proteomics to characterize cellular protein changes during HCMV latency and reactivation in T98G cells. A total of 168 differentially expressed proteins (DEPs) were identified, including 89 proteins in latency and 85 proteins in reactivation. Bioinformatics analysis showed that a few biological pathways were associated with HCMV latency or reactivation. Moreover, we validated 16 DEPs by both mRNA and protein expression profiles and further evaluated the effects of ApoE and PI3K pathway on HCMV infection. ApoE knockdown reduced HCMV loads and virus release, whereas overexpressing ApoE hampered HCMV latent infection, indicating a role in HCMV latency establishment/maintenance. Blocking the PI3K pathway by LY294002, a PI3K inhibitor, induced HCMV reactivation from latency in T98G cells. Overall, this comparative proteomic analysis delineates the cellular protein changes during HCMV latency and reactivation and provides a road map to advance our understanding of the mechanism(s) in the context of neural cells. IMPORTANCE Human cytomegalovirus (HCMV) is a highly transmissible beta-herpesvirus that has a prevalence of 60%-90% worldwide. This opportunist pathogen poses a significant threat to newborns and immunosuppressed individuals. One major obstacle for developing effective therapeutics is a poor understanding of HCMV latency/reactivation mechanisms. This study presents, for the first time, a systemic analysis of host cell protein expression changes during HCMV latency establishment and reactivation processes in neural cells. We showed that ApoE was downregulated by HCMV to facilitate latent infection. Also, the proteomic analysis has associated a few PI3K pathway-related proteins with HCMV reactivation. Altogether, this study highlights multiple host proteins and signaling pathways that can be further investigated as potential druggable targets for HCMV-related diseases, especially brain disorders.

Stringent Response in Mycobacteria: From Biology to Therapeutic Potential

Mycobacterium tuberculosis is a human pathogen that can thrive inside the host immune cells for several years and cause tuberculosis. This is due to the propensity of M. tuberculosis to synthesize a sturdy cell wall, shift metabolism and growth, secrete virulence factors to manipulate host immunity, and exhibit stringent response. These attributes help M. tuberculosis to manage the host response, and successfully establish and maintain an infection even under nutrient-deprived stress conditions for years. In this review, we will discuss the importance of mycobacterial stringent response under different stress conditions. The stringent response is mediated through small signaling molecules called alarmones “(pp)pGpp”. The synthesis and degradation of these alarmones in mycobacteria are mediated by Rel protein, which is both (p)ppGpp synthetase and hydrolase. Rel is important for all central dogma processes—DNA replication, transcription, and translation—in addition to regulating virulence, drug resistance, and biofilm formation. Rel also plays an important role in the latent infection of M. tuberculosis. Here, we have discussed the literature on alarmones and Rel proteins in mycobacteria and highlight that (p)ppGpp-analogs and Rel inhibitors could be designed and used as antimycobacterial compounds against M. tuberculosis and non-tuberculous mycobacterial infections.

1406. Hepatitis B and C Prevalence in Patients with Active and Latent Tuberculosis in an Ethnically Diverse Area of London, UK

Background North West London has one of the highest tuberculosis (TB) rates in the UK, at 24.8 per 10,000. The UK prevalence of hepatitis B virus (HBV) is 0.1-0.5% and for hepatitis C virus (HCV) is 0.5-1%. Chronic infection with HBV or HCV can lead to an increased risk of adverse treatment outcomes, such as drug-induced liver injury (DILI) in patients with active or latent TB. National guidelines recommend routinely screening for HBV/HCV prior to initiating TB treatment. Our objectives were to 1) evaluate the HBV/HCV screening practice in local TB clinics, 2) establish the prevalence of HBV/HCV in patients receiving TB treatment. Methods Retrospective analysis of laboratory and medical records of patients treated for active or latent TB identified from the London TB register and clinic records from 01/01/2018 to 31/12/2020 from London North West NHS Trust. Results 1409 patients received treatment for TB during the time period of interest; 574 (40.7%) had active disease and 835 (59.3%) had latent infection. 966/1409 patients (68.56%) were screened for HBV and HCV. 55.9% of the active TB group and 77.2% of the latent infection group were tested. 66 (6.8%) patients had isolated anti-HBc positivity, 22 (2.3%) were HBV surface antigen positive and 8 (0.8%) were HCV-antibody positive. HBV surface antigens were more prevalent in active TB patients: 9/321 (2.80%) with active TB versus 13/645 (2.02%) with latent TB. 36/321 (11.21%) active TB patients had HBV core antibodies compared to 30/645 (4.65%) latent TB patients (p < 0.001). Three patients started antiviral treatment following their viral hepatitis diagnosis (one with HBV, two with HCV). Conclusion The prevalence of chronic HBV in the study population was higher than the estimated UK prevalence. Fifteen diagnoses of hepatitis were new, allowing specialist referral for monitoring of fibrosis and development of hepatocellular carcinoma. Three patients required hepatitis treatment. 6.8% of patients were positive for anti-HBc and therefore identified as being at future risk of HBV reactivation if requiring immunosuppressive therapies.TB disproportionately affects marginalised communities; screening for viral hepatitis in TB clinic represents an opportunity to target these hard-to-reach groups to maximise the impact of public health interventions. Disclosures All Authors: No reported disclosures