G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival.

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that the coordinated action of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) orchestrate cholesterol build-up in in vitro and in vivo mouse models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while SIRT6 along with G9a represses the genes involved in cholesterol efflux. The accumulated cholesterol in Mtb infected macrophages promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and pharmacological inhibition in vivo; or utilization of BMDMs derived from Sirt6-/- mice or in vivo infection in haplo-insufficient Sirt6-/+ mice; hampered host cholesterol accumulation and restricted Mtb burden. These findings shed light on the novel roles of G9a and SIRT6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

Sirtuin 6 deficiency transcriptionally up-regulates TGF-ß signaling and induces fibrosis in mice.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform.

Tissue engineering approaches are used to mimic the microenvironment of the skeletal muscle in vitro. However, the validation of a bioengineered muscle as a model to study diseases is inadequate. Here, we present polycaprolactone nanofibers as a robust platform that mimics cellular organization and recapitulates critical functions of the myotubes observed in vivo. We isolated myoblasts from mice following a simplified protocol and cultured them on aligned nanofibers. Myotubes grown on aligned nanofibers maintained alignment for 14 days and exhibited a time-dependent increase in levels of p-AKT upon insulin stimulation. Treatment with matrix-assisted integrin inhibitor led to reduction in p-AKT levels, underscoring the critical role of environment on the biological processes. We demonstrate the suitability of myotubes grown on nanofibrous platform to study corticosteroid-induced muscle degeneration. This study, thus, demonstrates that aligned nanofibers retain myotubes in culture for longer duration and recapitulate the functions of skeletal muscle under pathophysiological conditions.

Toll-like receptor 2 deficiency hyperactivates the FoxO1 transcription factor and induces aging-associated cardiac dysfunction in mice.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors involved in innate immunity. Previous studies have shown that TLR2 inhibition protects the heart from acute stress, including myocardial infarction and doxorubicin-induced cardiotoxicity in animal models. However, the role of TLR2 in the development of aging-associated heart failure is not known. In this work, we studied aging-associated changes in structure and function of TLR2-deficient mice hearts. Whereas young TLR2-KO mice did not develop marked cardiac dysfunction, 8- and 12-month-old TLR2-KO mice exhibited spontaneous adverse cardiac remodeling and cardiac dysfunction in an age-dependent manner. The hearts of the 8-month-old TLR2-KO mice had increased fibrosis, cell death, and reactivation of fetal genes. Moreover, TLR2-KO hearts displayed reduced infiltration by macrophages, increased numbers of myofibroblasts and atrophic cardiomyocytes, and higher levels of the atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Mechanistically, TLR2 deficiency impaired the PI3K/Akt signaling pathway, leading to hyperactivation of the transcription factor Forkhead box protein O1 (FoxO1) and, in turn, to elevated expression of FoxO target genes involved in the regulation of muscle wasting and cell death. AS1842856-mediated chemical inhibition of FoxO1 reduced the expression of the atrophy-related ubiquitin ligases and significantly reversed the adverse cardiac remodeling while improving the contractile functions in the TLR2-KO mice. Interestingly, TLR2 levels decreased in hearts of older mice, and the activation of TLR1/2 signaling improved cardiac functions in these mice. These findings suggest that TLR2 signaling is essential for protecting the heart against aging-associated adverse remodeling and contractile dysfunction in mice.

Sirtuin 6 mediated stem cell cardiomyogenesis on protein coated nanofibrous scaffolds.

The cellular niche provides combination of biomolecular and biophysical cues to control stem cell fate. Three-dimensional (3D) aligned nanofibrous scaffolds can effectively augment stem cell cardiomyogenesis. This work aims to understand the role of biomolecular signals from extracellular matrix (ECM) proteins and leverage them to further promote cardiomyogenesis on nanofibrous scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on 3D aligned polycaprolactone scaffolds coated with different ECM proteins. Among multiple coatings tested, collagen coated fibers were most effective in promoting cardiomyogenesis as determined from increased expression of cardiac biomarkers and intracellular calcium flux. At molecular level, enhanced differentiation on collagen coated fibers was associated with an increased level of sirtuin 6 (SIRT6). Depletion of SIRT6 using siRNA attenuated the differentiation process through activation of Wnt signaling pathway. This study, thus, demonstrates that protein coated scaffolds can augment cardiomyogenic differentiation of stem cells through a combination of topographical and biomolecular signals.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart.

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.

Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and ß-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and ß-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

Regulation of Akt signaling by sirtuins: its implication in cardiac hypertrophy and aging.

Cardiac hypertrophy is a multifactorial disease characterized by multiple molecular alterations. One of these alterations is change in the activity of Akt, which plays a central role in regulating a variety of cellular processes ranging from cell survival to aging. Akt activation is mainly achieved by its binding to phosphatidylinositol (3,4,5)-triphosphate. This results in a conformational change that exposes the kinase domain of Akt for phosphorylation and activation by its upstream kinase, 3-phosphoinositide-dependent protein kinase 1, in the cell membrane. Recent studies have shown that sirtuin isoforms, silent information regulator (SIRT) 1, SIRT3, and SIRT6, play an essential role in the regulation of Akt activation. Although SIRT1 deacetylates Akt to promote phosphatidylinositol (3,4,5)-triphosphate binding and activation, SIRT3 controls reactive oxygen species-mediated Akt activation, and SIRT6 transcriptionally represses Akt at the level of chromatin. In the first part of this review, we discuss the mechanisms by which sirtuins regulate Akt activation and how they influence other post-translational modifications of Akt. In the latter part of the review, we summarize the implications of sirtuin-dependent regulation of Akt signaling in the control of major cellular processes such as cellular growth, angiogenesis, apoptosis, autophagy, and aging, which are involved in the initiation and progression of several diseases.

Deficiency of cardiomyocyte-specific microRNA-378 contributes to the development of cardiac fibrosis involving a transforming growth factor ß (TGFß1)-dependent paracrine mechanism.

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFß1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFß1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFß1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFß1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFß1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.

A cardiac-enriched microRNA, miR-378, blocks cardiac hypertrophy by targeting Ras signaling.

Understanding the regulation of cardiomyocyte growth is crucial for the management of adverse ventricular remodeling and heart failure. MicroRNA-378 (miR-378) is a newly described member of the cardiac-enriched miRNAs, which is expressed only in cardiac myocytes and not in cardiac fibroblasts. We have previously shown that miR-378 regulates cardiac growth during the postnatal period by direct targeting of IGF1R (Knezevic, I., Patel, A., Sundaresan, N. R., Gupta, M. P., Solaro, R. J., Nagalingam, R. S., and Gupta, M. (2012) J. Biol. Chem. 287, 12913-12926). Here, we report that miR-378 is an endogenous negative regulator of cardiac hypertrophy, and its levels are down-regulated during hypertrophic growth of the heart and during heart failure. In primary cultures of cardiomyocytes, overexpression of miR-378 blocked phenylephrine (PE)-stimulated Ras activity and also prevented activation of two major growth-promoting signaling pathways, PI3K-AKT and Raf1-MEK1-ERK1/2, acting downstream of Ras signaling. Overexpression of miR-378 suppressed PE-induced phosphorylation of S6 ribosomal kinase, pERK1/2, pAKT, pGSK-3ß, and nuclear accumulation of NFAT. There was also suppression of the fetal gene program that was induced by PE. Experiments carried out to delineate the mechanism behind the suppression of Ras, led us to identify Grb2, an upstream component of Ras signaling, as a bona fide direct target of miR-378-mediated regulation. Deficiency of miR-378 alone was sufficient to induce fetal gene expression, which was prevented by knocking down Grb2 expression and blocking Ras activation, thus suggesting that miR-378 interferes with Ras activation by targeting Grb2. Our study demonstrates that miR-378 is an endogenous negative regulator of Ras signaling and cardiac hypertrophy and its deficiency contributes to the development of cardiac hypertrophy.

Essential Role of Anisotropy in Bioengineered Cardiac Tissue Models.

As regulatory bodies encourage alternatives to animal testing, there is renewed interest in engineering disease models, particularly for cardiac tissues. The aligned organization of cells in the mammalian heart controls the electrical and ionic currents and its ability to efficiently circulate blood to the body. Although the development of engineered cardiac systems is rising, insights into the topographical aspects, in particular, the necessity to design in vitro cardiac models incorporating cues for unidirectional cell growth, is lacking. This review first summarizes the widely used methods to organize cardiomyocytes (CMs) unidirectionally and the ways to quantify the resulting cellular alignment. The behavior of CMs in response to alignment is described, with emphasis on their functions and underlying mechanisms. Lastly, the limitations of state-of-the-art techniques to modulate CM alignment in vitro and opportunities for further development in the future to improve the cardiac tissue models that more faithfully mimic the pathophysiological hallmarks are outlined. This review serves as a call to action for bioengineers to delve deeper into the in vivo role of cellular organization in cardiac muscle tissue and draw inspiration to effectively mimic in vitro for engineering reliable disease models.

Star-PAP controls oncogene expression through primary miRNA 3'-end formation to regulate cellular proliferation and tumour formation.

Star-PAP is a non-canonical poly(A) polymerase that is down regulated in breast cancer. While Star-PAP down regulation impairs target mRNA polyadenylation, paradoxically, we see up regulation of a large number of oncogenes on Star-PAP knockdown. Using two breast cancer cells (MCF7 with high Star-PAP, and MDA-MB-231 with negligible Star-PAP level), we discover that Star-PAP negatively regulates oncogene expression and subsequently cellular proliferation. This regulation is compromised with Star-PAP mutant of 3'-end processing function (serine 6 to alanine, S6A phospho-mutation). Concomitantly, xenograft mice model using MDA-MB-231 cells reveals a reduction in the tumour formation on ectopic Star-PAP expression that is ameliorated by S6A mutation. We find that Star-PAP control of target oncogene expression is independent of Star-PAP-mediated alternative polyadenylation or target mRNA 3'-end formation. We demonstrate that Star-PAP regulates target oncogenes through cellular miRNAs (miR-421, miR-335, miR-424, miR-543, miR-205, miR-34a, and miR-26a) that are down regulated in breast cancer. Analysis of various steps in miRNA biogenesis pathway reveals that Star-PAP regulates 3'-end formation and synthesis of primary miRNA (host) transcripts that is dependent on S6 phosphorylation thus controlling mature miRNA generation. Using mimics and inhibitors of two target miRNAs (miR-421 and miR-424) after Star-PAP depletion in MCF7 or ectopic expression in MDA-MB-231 cells, we demonstrate that Star-PAP controls oncogene expression and cellular proliferation through targeting miRNAs that regulates tumour formation. Our study establishes a novel mechanism of oncogene expression independent of alternative polyadenylation through Star-PAP-mediated miRNA host transcript polyadenylation that regulates breast cancer progression.

Proto-oncogene cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program in the heart and controls cardiac hypertrophy.

AIMS: RBM10 is a well-known RNA binding protein that regulates alternative splicing in various disease states. We have shown a splicing-independent function of RBM10 that regulates heart failure. This study aims to unravel a new biological function of RBM10 phosphorylation by proto-oncogene cSrc that enables anti-hypertrophy gene program and controls cardiac hypertrophy. MATERIALS AND METHODS: We employ in vitro and in vivo approaches to characterise RBM10 phosphorylation at three-tyrosine residues (Y81, Y500, and Y971) by cSrc and target mRNA regulation. We also use isoproterenol induced rat heart and cellular hypertrophy model to determine role of cSrc-mediated RBM10 phosphorylation. KEY FINDINGS: We show that RBM10 phosphorylation is induced in cellular and animal heart model of cardiac hypertrophy and regulates target mRNA expression and 3'-end formation. Inhibition of cSrc kinase or mutation of the three-tyrosine phosphorylation sites to phenylalanine accentuates myocyte hypertrophy, and results in advancement and an early attainment of hypertrophy in the heart. RBM10 is down regulated in the hypertrophic myocyte and that its re-expression reverses cellular and molecular changes in the myocyte. However, in the absence of phosphorylation (cSrc inhibition or phospho-deficient mutation), restoration of endogenous RBM10 level in the hypertrophic heart or ectopic re-expression in vitro failed to reverse cardiomyocyte hypertrophy. Mechanistically, loss of RBM10 phosphorylation inhibits nuclear localisation and interaction with Star-PAP compromising anti-hypertrophy gene expression. SIGNIFICANCE: Our study establishes that cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program. We also report a new functional regulation of RBM10 by phosphorylation that is poised to control heart failure.

A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival.

Postnatal cardiac remodeling is characterized by a marked decrease in the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) expression. The underlying mechanism remains unexplored. This study examined the role of microRNAs in postnatal cardiac remodeling. By expression profiling, we observed a 10-fold increase in miR-378 expression in 1-week-old neonatal mouse hearts compared with 16-day-old fetal hearts. There was also a 4-6-fold induction in expression of miR-378 in older (10 months) compared with younger (1 month) hearts. Interestingly, tissue distribution analysis identified miR-378 to be highly abundant in heart and skeletal muscles. In the heart, specific expression was observed in cardiac myocytes, which was inducible by a variety of stressors. Overexpression of miR-378 enhanced apoptosis of cardiomyocytes by direct targeting of IGF1R and reduced signaling in Akt cascade. The inhibition of miR-378 by its anti-miR protected cardiomyocytes against H(2)O(2) and hypoxia reoxygenation-induced cell death by promoting IGF1R expression and downstream Akt signaling cascade. Additionally, our data show that miR-378 expression is inhibited by IGF1 in cardiomyocytes. In tissues such as fibroblasts and fetal hearts, where IGF1 levels are high, we found either absent or significantly low miR-378 levels, suggesting an inverse relationship between these two factors. Our study identifies miR-378 as a new cardioabundant microRNA that targets IGF1R. We also demonstrate the existence of a negative feedback loop between miR-378, IGF1R, and IGF1 that is associated with postnatal cardiac remodeling and with the regulation of cardiomyocyte survival during stress.

Nampt secreted from cardiomyocytes promotes development of cardiac hypertrophy and adverse ventricular remodeling.

Nicotinamide phosphoribosyltransferase (Nampt) is an important coenzyme involved in cellular redox reactions. Inside the cell, Nampt (iNampt) functions as a rate-limiting enzyme in the NAD salvage pathway, and outside the cell (eNampt), it acts as a proinflammatory cytokine. High-circulating levels of Nampt are reported in different pathological conditions. This study was designed to examine the role of Nampt in the development of cardiac hypertrophy and ventricular remodeling. We studied the hypertrophic response in Nampt heterozygous (+/-) knockout and cardiac-specific overexpressing Nampt transgenic mice. Whereas Nampt(+/-) mice were protected against agonist (isoproterenol and angiotensin II)-induced hypertrophy, Nampt transgenic mice spontaneously developed cardiac hypertrophy at 6 mo of age. Experiments conducted to gain insight into the mechanism revealed that treatment of cardiomyocytes with recombinant (eNampt) or overexpression with Nampt-synthesizing adenovirus vector (Ad.Nampt) induced cardiomyocyte hypertrophy. The prohypertrophic effects of eNampt and Ad.Nampt were blocked by the addition of a Nampt-blocking antibody into cultures, thus suggesting that Nampt was in fact invoking hypertrophic response of cardiomyocytes by acting on the cell surface receptors. We also found increased Nampt levels in the supernatant of cardiomyocyte cultures subjected to stress by either serum starvation or H(2)O(2) treatment. Exploration of signaling pathways in Nampt-induced cardiac hypertrophy and fibrosis revealed increased activation of mitogen-activated protein kinases, namely, JNK1, p38, and ERK. This was also associated with increased calcineurin levels and nuclear factor of activated T-cell localization into the nucleus. From these studies we conclude that cardiomyocytes are capable of secreting Nampt during stress, and exogenous Nampt is a positive regulator of cardiac hypertrophy and adverse ventricular remodeling.

Unique Tandem Repeats in the Inverted Terminal Repeat Regions of Monkeypox Viruses.

The genetic diversity, especially in noncoding regions between clade I, clade IIa, and clade IIb monkeypox viruses (MPXVs), is still not fully understood. Here, we report that unique 16-nucleotide-length tandem repeats in MPXVs viruses are located in the noncoding regions of inverted terminal repeats (ITR), and the copy number of this repeat is different among clade I, clade IIa, and clade IIb viruses. It is noteworthy that tandem repeats containing these specific sequences (AACTAACTTATGACTT) are only present in MPXVs and are not found in other poxviruses. Also, the tandem repeats containing these specific sequences (AACTAACTTATGACTT) do not correspond to the tandem repeats present in the human and rodent (mice and rat) genomes. On the other hand, some of the reported tandem repeats in the human and rodent (mice and rat) genomes are present in the clade IIb-B.1 lineage of MPXV. In addition, it is noteworthy that the genes flanking these tandem repeats are lost and gained compared between clade I, clade IIa, and clade IIb MPXV. IMPORTANCE The different groups of MPXVs contain unique tandem repeats with different copy numbers in the ITR regions, and these repeats may be likely to play a role in the genetic diversity of the virus. Clade IIb (B) MPXV contains 38 and 32 repeats similar to the Tandem repeats reported in the human and rodent genome, respectively. However, none of these 38 (human) and 32 (rodent) tandem repeats matched the tandem repeats (AACTAACTTATGACTT) found in the present study. Finally, when developing attenuated or modified MPXV vaccine strains, these repeats in noncoding genomic regions can be exploited to incorporate foreign proteins (adjuvants/other virus proteins/racking fluorescent proteins such as green fluorescent protein) to carry out studies such as vaccine production and virus pathogenesis.

Deciphering the Origin of DNA Viruses (Replication-Associated Parvo-NS1) That Infect Vertebrates from Invertebrate-Infecting Viruses.

DNA replication is a standard and essential function among DNA viruses; however, this functional domain's common ancestor, origin, and evolutionary path in invertebrate- and vertebrate-infecting viruses are not yet fully understood. Here, we present evidence, using a combination of phylogenetic relationships, coevolution, and CLANS (cluster analysis of sequences) analysis, that the parvo-NS1 domain (nonstructural protein NS1, DNA helicase domain) of these DNA viruses that infect vertebrates potentially originated from the invertebrate (Platyhelminthes) parvo-NS1 domain of parvovirus-related sequences (PRSs). Our results suggest that papillomaviruses and the parvovirus subfamilies Densovirinae and Hamaparvovirinae DNA helicase evolved directly from the Platyhelminthes NS1 domain (PRSs). Similarly, the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the PRSs through Hamaparvovirinae. Further, our analysis also clarified that herpesviruses and adenoviruses independently obtained the parvo-NS1 domain from Dependoparvovirus (Parvovirinae). Furthermore, virus-host coevolution analysis revealed that the parvovirus NS1 domain has coevolved with hosts, from flatworms to humans, and it appears that the papillomavirus may have obtained the DNA helicase during the early stages of parvovirus evolution and later led to the development of the DNA helicase of adomavirus and polyomavirus. Finally, herpesviruses and adenoviruses likely inherited the parvo-NS1 domain from Dependoparvovirus in the later stages of evolution. To the best of our knowledge, this is the first evolutionary evidence to suggest that the DNA helicase of viruses that infect vertebrates originated from the invertebrate PRSs. IMPORTANCE DNA replication of DNA viruses is an essential function. This allows DNA replication of viruses to form virus particles. The DNA helicase domain is responsible for this primary function. This domain is present in parvoviruses, papillomaviruses, polyomaviruses, herpesviruses, and adenoviruses. But little is known about the common ancestor, origin, and evolutionary path of DNA helicase in invertebrate- and vertebrate-infecting viruses. Here, we report the possibility of the origin of DNA viruses (DNA helicase) infecting vertebrates from Platyhelminthes (invertebrate) PRSs. Our study established that the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the Platyhelminthes PRSs through Hamaparvovirinae. Furthermore, our study suggests that the papillomavirus DNA helicase may have evolved in the early stages of parvovirus evolution and then led to the development of the adomavirus and polyomavirus. Our study suggests that the herpesviruses and adenoviruses likely inherited the parvo-NS1 domain through gene capture from Dependoparvovirus in the later stages of parvovirus evolution in their hosts.