Viral Gene Products Research Articles

Engineered Resistance to Tobamoviruses.

Tobacco mosaic virus (TMV) was the first virus to be studied in detail and, for many years, TMV and other tobamoviruses, particularly tomato mosaic virus (ToMV) and tobamoviruses infecting pepper (Capsicum spp.), were serious crop pathogens. By the end of the twentieth and for the first decade of the twenty-first century, tobamoviruses were under some degree of control due to introgression of resistance genes into commercial tomato and pepper lines. However, tobamoviruses remained important models for molecular biology, biotechnology and bio-nanotechnology. Recently, tobamoviruses have again become serious crop pathogens due to the advent of tomato brown rugose fruit virus, which overcomes tomato resistance against TMV and ToMV, and the slow but apparently inexorable worldwide spread of cucumber green mottle mosaic virus, which threatens all cucurbit crops. This review discusses a range of mainly molecular biology-based approaches for protecting crops against tobamoviruses. These include cross-protection (using mild tobamovirus strains to 'immunize' plants against severe strains), expressing viral gene products in transgenic plants to inhibit the viral infection cycle, inducing RNA silencing against tobamoviruses by expressing virus-derived RNA sequences in planta or by direct application of double-stranded RNA molecules to non-engineered plants, gene editing of host susceptibility factors, and the transfer and optimization of natural resistance genes.

TRIM5α: A Protean Architect of Viral Recognition and Innate Immunity.

The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided 'specificity' conferred by the totality of TRIM5α's additional domains that may account for its recently identified promiscuity.

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

Sp1 facilitates continued HSV-1 gene expression in the absence of key viral transactivators.

Productive replication of herpes simplex virus (HSV) relies upon a well-ordered transcriptional cascade flowing from immediate-early (IE) to early (E) to late (L) gene products. While several virus-encoded transcriptional activators are involved in this process, IE and E gene promoters also contain multiple binding sites for the ubiquitously expressed cellular transcription factor Sp1. Sp1 has been previously implicated in activating HSV-1 gene transcription downstream of these sites, but why Sp1-binding sites are maintained in the promoters of genes activated by virus-encoded activators remains unclear. We hypothesized that Sp1 enables continued HSV-1 transcription and replication when viral transactivators are limited. We used a depletion-based approach in human foreskin fibroblasts to investigate the specific contribution of Sp1 to the initiation and progression of the HSV-1 lytic gene cascade. We found that Sp1 increased viral transcript levels, protein expression, and replication following infection with VP16- or ICP0-deficient viruses but had little to no effect on rescued viruses or during wild-type (WT) HSV-1 infection. Moreover, Sp1 promoted WT virus transcription and replication following interferon treatment of fibroblasts and thus may contribute to viral immune evasion. Interestingly, we observed reduced expression of Sp1 and Sp1-family transcription factors in differentiated sensory neurons compared to undifferentiated cells, suggesting that reduced Sp1 levels may also contribute to HSV-1 latent infection. Overall, these findings indicate that Sp1 can promote HSV-1 gene expression in the absence of key viral transactivators; thus, HSV-1 may use Sp1 to maintain its gene expression and replication under adverse conditions.IMPORTANCEHerpes simplex virus (HSV) is a common human pathogen that actively replicates in the epithelia but can persist for the lifetime of the infected host via a stable, latent infection in neurons. A key feature of the HSV replication cycle is a complex transcriptional program in which virus and host-cell factors coordinate to regulate expression of the viral gene products necessary for continued viral replication. Multiple binding sites for the cellular transcription factor Sp1 are located in the promoters of HSV-1 genes, but how Sp1 binding contributes to transcription and replication of wild-type virus is not fully understood. In this study, we identified a specific role for Sp1 in maintaining HSV-1 gene transcription under adverse conditions, as when virus-encoded transcriptional activators were absent or limited. Preservation of Sp1-binding sites in HSV-1 gene promoters may thus benefit the virus as it navigates diverse cell types and host-cell conditions during infection.

Prunella vulgaris polysaccharide inhibits herpes simplex virus infection by blocking TLR-mediated NF-κB activation

BackgroundPrunella vulgaris polysaccharide extracted by hot water and 30% ethanol precipitation (PVE30) was reported to possess potent antiviral effects against herpes simplex virus (HSV) infection. However, its anti-HSV mechanism has not yet been fully elucidated.PurposeThis study aimed to investigate the potential mechanisms of PVE30 against HSV infection.MethodsAntiviral activity was evaluated by a plaque reduction assay, and the EC50 value was calculated. Immunofluorescence staining and heparin bead pull-down assays confirmed the interactions between PVE30 and viral glycoproteins. Real-time PCR was conducted to determine the mRNA levels of viral genes, including UL54, UL29, UL27, UL44, and US6, and the proinflammatory cytokines IL-6 and TNF-α. The protein expression of viral proteins (ICP27, ICP8, gB, gC, and gD), the activity of the TLR-NF-κB signalling pathway, and necroptotic-associated proteins were evaluated by Western blotting. The proportion of necroptotic cells was determined by flow cytometric analysis.ResultsThe P. vulgaris polysaccharide PVE30 was shown to compete with heparan sulfate for interaction with HSV surface glycoprotein B and gC, thus strongly inhibiting HSV attachment to cells. In addition, PVE30 downregulated the expression of IE genes, which subsequently downregulated the expression of E and L viral gene products, and thus effectively restricted the yield of progeny virus. Further investigation confirmed that PVE30 inhibited TLR2 and TLR3 signalling, leading to the effective suppression of NF-κB activation and IL-6 and TNF-α expression levels, and blocked HSV-1-induced necroptosis by reducing HSV-1-induced phosphorylation of MLKL.ConclusionOur results demonstrate that the P. vulgaris polysaccharide PVE30 is a potent anti-HSV agent that blocks TLR-mediated NF-κB activation.Graphical

Uncloaking the viral glycocalyx: How do viruses exploit glycoimmune checkpoints?

The surfaces of cells and enveloped viruses alike are coated in carbohydrates that play multifarious roles in infection and immunity. Organisms across all kingdoms of life make use of a diverse set of monosaccharide subunits, glycosidic linkages, and branching patterns to encode information within glycans. Accordingly, sugar-patterning enzymes and glycan binding proteins play integral roles in cell and organismal biology, ranging from glycoprotein quality control within the endoplasmic reticulum to lymphocyte migration, coagulation, inflammation, and tissue homeostasis. Unsurprisingly, genes involved in generating and recognizing oligosaccharide patterns are playgrounds for evolutionary conflicts that abound in cross-species interactions, exemplified by the myriad plant lectins that function as toxins. In vertebrates, glycans bearing acidic nine-carbon sugars called sialic acids are key regulators of immune responses. Various bacterial and fungal pathogens adorn their cells in sialic acids that either mimic their hosts' or are stolen from them. Yet, how viruses commandeer host sugar-patterning enzymes to thwart immune responses remains poorly studied. Here, we review examples of viruses that interact with sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immune cell receptors that regulate toll-like receptor signaling and govern glycoimmune checkpoints, while highlighting knowledge gaps that merit investigation. Efforts to illuminate how viruses leverage glycan-dependent checkpoints may translate into new clinical treatments that uncloak viral antigens and infected cell surfaces by removing or masking immunosuppressive sialoglycans, or by inhibiting viral gene products that induce their biosynthesis. Such approaches may hold the potential to unleash the immune system to clear long intractable chronic viral infections.

High Levels of Monocyte Chemoattractant Protein-1 Plasma Are A Risk Factor for Neurocognitive Disorders in Human Immunodeficiency Virus Infection with Antiretroviral Therapy

Background: HIV-associated neurocognitive disorder (HAND) is a neurocognitive dysfunction caused by HIV infection. The prevalence of neurocognitive disorders of HIV in Indonesia is about 51%. Despite receiving antiretroviral therapy, HAND is still a common complication that causes morbidity in people with HIV. One of the foundations of HAND pathogenesis is inflammation. Infection of macrophages/microglia and astrocytes leads to excessive production of viral gene products and chemokines, one of them is Monocyte chemoattractant protein 1 (MCP-1). The purpose of this study was to determine plasma MCP-1 levels that increase the risk of neurocognitive disorders in HIV infection with antiretroviral therapy. Methods: This matched case-control study involved 76 subjects with HIV, 38 subjects with neurocognitive disorders, and 38 subjects without neurocognitive disorders. Cognitive status was reviewed based on MoCA-Ina examination criteria. Plasma MCP-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: Of the 76 subjects, the mean age of the case group was 42.08 ± 9.44 years and the control group was 42.16± 8.90 years. The cut point value based on the ROC curve of the plasma MCP-1 level was 82.14 pg/mL. It had an 86.8% sensitivity and 52.6% specificity for the occurrence of neurocognitive disorders in HIV. The risk of neurocognitive disorders in HIV was found in MCP-1 plasma ≥ 82.14 pg/mL (OR 7.33, p < 0.001, CI 95% = 2.35-22.83). Based on multivariate analysis, MCP-1 levels were independently associated with the incidence of neurocognitive disorders in HIV (AOR 11.01, CI 95% = 2.75-44.01, p=0.001). Conclusion: This study showed that high plasma MCP-1 levels were an event risk factor for neurocognitive disorders in HIV with antiretroviral therapy.

Effective Agrobacterium-mediated genetic transformation of okra (Abelmoschus esculentus L.) and generation of RNAi plants resistant to Begomovirus infecting okra

ABSTRACT Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture–dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene–specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture–mediated RNAi-derived resistance in okra against Begomovirus infection.

Cytomegalovirus infection induces Alzheimer's disease-associated alterations in tau.

Alzheimer's disease (AD) manifests with loss of neurons correlated with intercellular deposition of amyloid (amyloid plaques) and intracellular neurofibrillary tangles of hyperphosphorylated tau. However, targeting AD hallmarks has not as yet led to development of an effective treatment despite numerous clinical trials. A better understanding of the early stages of neurodegeneration may lead to development of more effective treatments. One underexplored area is the clinical correlation between infection with herpesviruses and increased risk of AD. We hypothesized that similar to work performed with herpes simplex virus 1 (HSV1), infection with the cytomegalovirus (CMV) herpesvirus increases levels and phosphorylation of tau, similar to AD tauopathy. We used murine CMV (MCMV) to infect mouse fibroblasts and rat neuronal cells to test our hypothesis. MCMV infection increased steady-state levels of primarily high molecular weight forms of tau and altered the patterns of tau phosphorylation. Both changes required viral late gene products. Glycogen synthase kinase 3 beta (GSK3β) was upregulated in the HSVI model, but inhibition with lithium chloride suggested that this enzyme is unlikely to be involved in MCMV infection mediated tau phosphorylation. Thus, we confirm that MCMV, a beta herpes virus, like alpha herpes viruses (e.g., HSV1), can promote tau pathology. This suggests that CMV infection can be useful as another model system to study mechanisms leading to neurodegeneration. Since MCMV infects both mice and rats as permissive hosts, our findings from tissue culture can likely be applied to a variety of AD models to study development of abnormal tau pathology.

Viral DNA Accumulation Regulates Replication Efficiency of Chlorovirus OSy-NE5 in Two Closely Related Chlorella variabilis Strains.

Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus.

Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

The effect of virus-induced cellular transformation on oncogenesis

Aim to summarize the scientific data presented in the recent publications on tumor-associated processes induced by viruses. We analyzed 23 international publications devoted to the development and course of tumor-related processes associated with oncogenic viruses.
 The tumor-associated mechanisms are based on the processes of cell transformation, which largely depend on the state of telomeres. No less important are viral and cellular oncogenes, molecular circuits that control cell proliferation. Viral oncogenes encode proteins that increase the concentration of telomerase in the infected cells, and thereby increase the number of cell division cycles. The immune homeostasis, maintaining the integrity of body tissues, is regulated by activating and inhibiting metabolic pathways. The errors in the functioning of these signaling pathways caused by oncogenic viruses can lead to cell transformation and oncogenesis. Guaninenucleotide-binding protein RAS and protein kinase AKT are important components of signaling pathways that contribute to the production of D-type cyclins that control the cell cycle and regulate the activity of metabolic enzymes. Cyclin-dependent kinase is an important factor controlling cell cycles, damage and problems with nucleic acid replication, as well as proper assembly of the mitotic spindle. These processes can be disrupted by the transformation caused by oncogenic viruses. In most cases, viral oncogenes undergo additional changes that contribute to their transformation potential. The transformative activity of viral gene products correlates with binding to specific cellular proteins. In the immunopathogenesis of oncogenesis, an important role belongs to the inactivation of tumor suppressors, and the processes of phosphorylation.

Mycoviruses in Fungi: Carcinogenesis of Fungal Agents May Not Always Be Mycotoxin Related

Certain viruses have been found to induce diverse biological pathways to carcinogenesis, evidenced by the presence of viral gene products in some tumors. Despite the fact that many fungal agents contain mycoviruses, until recently, their possible direct effects on human health, including carcinogenesis and leukemogenesis, had not been explored. In this regard, most studies of fungal agents have rightly concentrated on their mycotoxin formation and effects. Recently, the direct role of yeasts and fungi in the etiology of cancers, including leukemia, have been investigated. While greater attention has been placed on the carcinogenic effects of Candida, the role of filamentous fungi in carcinogenesis has also been explored. Recent findings from studies using the enzyme-linked immunosorbent assay (ELISA) technique indicate that the plasma of patients with acute lymphoblastic leukemia (ALL) uniformly contains antibodies for a certain mycovirus-containing Aspergillus flavus, while controls are negative. The exposure of mononuclear leukocytes from patients with ALL in full remission, and long-term survivors, to the product of this organism was reported to result in the re-development of typical genetics and cell surface phenotypes characteristic of active ALL. Mycoviruses are known to be able to significantly alter the biological characteristics and functions of their host. The possible carcinogenic and leukemogenic role of mycoviruses, with and without their host, needs to be further investigated.

EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies.

There is increasing evidence that put into question the classical dogma that the Epstein-Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a third state has now been described, known as the abortive-lytic phase, which is characterized by the expression of some immediate early (IE) and early (E) genes, but no new virus progeny is produced. While the function of these IE and E gene products is not well understood, several recent studies support the concept they may contribute to tumor promotion by altering the tumor microenvironment (TME). The mechanisms by which these viral gene products may contribute to tumorigenesis remain unclear; however, it has been proposed that some of them promote cellular growth, immune evasion, and/or inhibit apoptosis. One of these EBV early gene products is the deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by BLLF3, which not only contributes to the establishment of latency through the production of activin A and IL-21, but it may also alter the TME, thus promoting oncogenesis.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

Human cytomegalovirus UL24 and UL43 products participate in SAMHD1 subcellular localization.

The online version contains supplementary material available at 10.1007/s13337-022-00799-3.

ODP564 Hypercalcemia in the Setting of HTLV-1 Infection and Normal PTHrP Levels

Abstract A 53 year old male with history of HTLV-1 infection associated myelopathy but with unknown mode of transmission, presented with acute on chronic bilateral lower extremity weakness and numbness with no other deficits. Examination revealed decreased strength (grade 1/5) in lower extremities with normal bulk, tone and sensations. Initial blood work revealed hypercalcemia with corrected Calcium of 16.2mg/dl(8.5-11.5), Phosphorus of 2.7mg/dl (2.5- 4.5) and Alkaline Phosphatase of 126mg/dl(20-140). Workup for hypercalcemia revealed Parathyroid hormone (PTH) level of 14pg/ml (10-65), 25 hydroxy vitamin D at 19.6 ng/ml(30-100), 1,25 dihydroxy vitamin D at 6.7pg/ml(19.9-79.3) and TSH of 1.265 microIU/ml(0.5-5). The Parathyroid hormone related protein(PTHrP) level was low at < 2pmol/L while Lactate Dehydrogenase (LDH) was 433U/L(100-220). Urine calcium creatinine ratio was 0.388. Reverse Transcriptase PCR was positive for HTLV-1 but negative for HTLV-2. Smudge cells and atypical lymphocytes were noted on peripheral smear. Imaging revealed generalized lymphadenopathy with splenomegaly but no spinal cord compression. Flow cytometry showed 7.8% atypical helper T cells, positive for CD3, CD4, CD5 and CD45. Clonal T cell receptor gene rearrangements were found, suggesting an underlying T cell neoplasm. Lymph node biopsy confirmed ATLL. He received treatment with fluids, Calcitonin and Denosumab after which serum Calcium levels fell (nadir 7.7mg/dl) and then normalized. He developed another episode of hypercalcemia 5 weeks later which was treated similarly. Discussion Human T cell Lymphotropic Virus-1 (HTLV-1) is a retrovirus causing asymptomatic infections, symptomatic systemic disorders and adult T cell Leukemia and lymphoma(ATLL). HTLV-1 infections, whether in asymptomatic or symptomatic patients, and ATLL are rare but important causes of hypercalcemia and hypercalcemic crises. Individuals with ATLL have the highest incidence of hypercalcemia(50-70%) among all hematologic malignancies. Hypercalcemia is a poor prognostic factor and predicts lower overall survival. It is caused by increased bone resorption, mediated by various factors with the largest role being played by Receptor Activator of Nuclear Factor Kappa B ligand (RANKL), followed by PTHrP, Interleukins, Macrophage Inflammatory Protein 1 alpha and gp 46. RANKL expression is increased upon transactivation by the viral TAX gene product and mediates conversion of osteoclast precursors to mature osteoclasts engaging in bone resorption. PTHrP expression is variable among patients, relating to levels of the TAX gene product that transactivates the PTHrP gene promoter. Thus, a low level, like in our patient, does not rule out HTLV infection as the cause of hypercalcemia. Management of hypercalcemia is in keeping with guidelines for management of other causes of hypercalcemia but may be better responsive to monoclonal antibodies against RANKL than bisphosphonates given the role of the ligand in mediating hypercalcemia in these cases. It can also be used in the setting of poor renal function, which is commonly encountered in hypercalcemic patients. Presentation: No date and time listed

Structural characterization of a soil viral auxiliary metabolic gene product – a functional chitosanase

Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins. We focus on AMGs that potentially encode chitosanase enzymes that metabolize chitin – a common carbon polymer. We express and functionally screen several chitosanase genes identified from environmental metagenomes. One expressed protein showing endo-chitosanase activity (V-Csn) is crystalized and structurally characterized at ultra-high resolution, thus representing the structure of a soil viral AMG product. This structure provides details about the active site, and together with structure models determined using AlphaFold, facilitates understanding of substrate specificity and enzyme mechanism. Our findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

HDAC inhibitors Panobinostat and Romidepsin enhance tax transcription in HTLV-1-infected cell lines and freshly isolated patients' T-cells.

The viral transactivator Tax plays a key role in HTLV-1 reactivation and de novo infection. Previous approaches focused on the histone deacetylase inhibitor (HDACi) Valproate as a latency-reversing agent to boost Tax expression and expose infected cells to the host’s immune response. However, following treatment with Valproate proviral load decreases in patients with HAM/TSP were only transient. Here, we hypothesize that other compounds, including more potent and selective HDACi, might prove superior to Valproate in manipulating Tax expression. Thus, a panel of HDACi (Vorinostat/SAHA/Zolinza, Panobinostat/LBH589/Farydak, Belinostat/PXD101/Beleodaq, Valproate, Entinostat/MS-275, Romidepsin/FK228/Istodax, and MC1568) was selected and tested for toxicity and potency in enhancing Tax expression. The impact of the compounds was evaluated in different model systems, including transiently transfected T-cells, chronically HTLV-1-infected T-cell lines, and freshly isolated PBMCs from HTLV-1 carriers ex vivo. We identified the pan-HDACi Panobinostat and class I HDACi Romidepsin as particularly potent agents at raising Tax expression. qRT-PCR analysis revealed that these inhibitors considerably boost tax and Tax-target gene transcription. However, despite this significant increase in tax transcription and histone acetylation, protein levels of Tax were only moderately enhanced. In conclusion, these data demonstrate the ability of Panobinostat and Romidepsin to manipulate Tax expression and provide a foundation for further research into eliminating latently infected cells. These findings also contribute to a better understanding of conditions limiting transcription and translation of viral gene products.