Emetine is a potent antiviral that acts on many viruses in the low-nM range, with several studies in animals and humans demonstrating antiviral activity. Historically, emetine was used to treat patients with Spanish influenza, in the last stages of the pandemic in the early 1900s. Some of these patients were “black” with cyanosis. Emetine rapidly reversed the cyanosis and other symptoms of this disease in 12–24 h. However, emetine also has been shown to have anti-inflammatory properties and it appears it is these anti-inflammatory properties that were responsible for the effects seen in patients with Spanish influenza. Emetine, in the past, has also been used in 10s to 100s of millions of people at a dose of ~60 mg daily to treat amoebiasis. Based on viral inhibition data we can calculate a likely SARS-CoV2 antiviral dose of ~1/10th the amoebiasis dose, which should dramatically reduce the risk of any side effects. While there are no anti-inflammatory dose response data available, based on the potential mode of action, the anti-inflammatory actions may also occur at low doses. This paper also examines the toxicity of emetine seen in clinical practice and that seen in the laboratory, and discusses the methods of administration aimed at reducing side effects if higher doses were found to be necessary. While emetine is a “pure drug” as it is extracted from ipecac, some of the differences between emetine and ipecac are also discussed. As discussed in the We previously discussed the potential use of emetine for the treatment of the SARS-CoV2 coronavirus, at 1/5th to 1/10th the amoebiasis treatment dose ,4, with Emetine was an important and effective anti-infective drug for the treatment of intestinal and extraintestinal amoebiasis, between the mid-1910s to the 1970s. It was likely administered to 10s–100s of millions of people in the treatment of amoebiasis see . It was Cephaëlis acuminata, or of Cephaëlis ipecacuanha [Ipecac Oral Solution” (Syrup of Ipecac) can range from an amount equal to, to an amount not more than 2.5 times, the content of emetine [75 grains of ipecacuanha, in sixteen and a half hours, without the slightest unpleasant effect on the patient, who had been unable to retain 1 grain (of ipecacuanha) when administered by mouth” [However, emetine is not ipecac. Ipecac consists of the dried rhizome and roots of cacuanha ,10. Thercacuanha . Emetinecacuanha ,13,14. Ecacuanha . The Uni emetine . If thesy mouth” .Emetine was shown to have significant antiviral and anti-inflammatory properties , and has50 values in the sub-µM and low-nM ranges, see 50) of mouse-adapted Ebola virus [50 of enterovirus, EV-A71, with the emetine group showing a million-fold reduction in viral titers in the fore and hind limbs [There is no question that emetine has potent antiviral actions, as exhibited by the numerous in vitro studies on multiple viruses demonstrating ECla virus  and all nd limbs . As expeCommon structural features used to classify viruses, such as enveloped, non-enveloped, DNA or RNA genome viruses, do not appear to affect sensitivity to emetine. Coronavirus, Rift Valley fever, Dengue, Zika and Ebola viruses are all enveloped viruses ,23,24,25Hanisch 1963  treated immediate favorable effects on appetite and general good feeling” [From 1938 to 1967 over 600 cases of viral hepatitis in children aged between 1 and 12 years old were treated with emetine at a dose of 1 mg/kg/day. In 100% of cases clinical cure was obtained between 7 and 14 days. No cases had prolonged illness or relapsed. None of the patients experienced undesirable side effects, but patients had “feeling” . In the early 1960s, Dr Antonio Fusillo an Italian physician, noticed rapid regression of zoster vesicles in patients being treated with emetine for amoebic dysentery. Through several animal experiments over time he determined that only very low doses of emetine, compared to that used for amoebic dysentery, where required to be an effective antiviral. ,45. Overextreme cyanosis, amounting to almost blackness”. Points’ description of his first patient where he noticed the “antiviral” activity of emetine was:During the two Spanish flu epidemics in New Orleans, in October 1918 and January 1919, Points treated 433 cases of influenza and 73 cases of flu pneumonia . One feaAlong about January 20 (1919), I treated a case of flu pneumonia complicated by a ruptured appendix. The patient developed acute nephritis, ran 20 per cent of albumen and passed large quantities of blood from the kidneys. Having had some previous experience with intravenous injections of emetine in renal hemorrhages, I injected 1/2 grain (~30 mg) emetine hydrochloride into his veins. I repeated this dose in twelve hours and the effect was marvelous, the patient’s temperature dropped from 104° to 101°, the hemorrhage ceased, his cough became better, and his pulse slowed down. The following morning, patient’s temperature was normal and his general condition much improved. Two injections were given that day with the results that patient’s cough subsided, the congestion of the kidneys passed off, the albumen disappeared, his pulse returned to normal and he seemed cured of his toxemia”“Points went on to describe the treatment of a group of patients:In the Emetine group I treated 65 cases 9 of which had pneumonia and 2 of which died. All of these nine cases of pneumonia were given from one to two intravenous injections of emetine per day. Purposely no quinin and aspirin were given and no antipyretic used. The result was the immediate lowering of the temperature from “...... the temperature from 104° to 103° (ranging between 103°–104°) to normal in from 12 to 24 hours, according as one or two doses of emetine per day were given. The temperature did not rise again and the disease was under control from that time on …… Several of these cases had edema of the lungs and were cyanosed until they were black …… but the results after the intravenous injections of emetine were astonishing. The temperature became normal, the cough subsided, the cyanosis disappeared, and the fight was won …… (he describes other treatment used) …… But the remarkable result was the rapid control of the temperature and the toxemia, and the rapid clearing up of the whole picture after the use of emetine”“However, there are hints that the response seen with emetine for the Spanish influenza virus, was not due to an antiviral effect, but possibly through acting on the inflammation caused by the virus and not the virus itself:I was so encouraged by the good effects of emetine, when given early enough, that I resolved to use it in all the flu cases, the drug once a day to seventeen simple, uncomplicated cases with these results: The emetine had no effect whatsoever on the non-complicated cases of influenza, not only not reducing the fever, but not controlling any of the symptoms. The disease in these cases ran a course of 3 to 5 days. But none of these cases developed any complications whatsoever from the flu, whereas some of the simple cases, that at first refused the intravenous injections of emetine, later on developed pneumonia and were saved by the drug. One of these simple cases, that refused the injections developed a good attack of cholecystitis, which was instantly relieved by the emetine treatment”“If this effect was truly an anti-inflammatory effect, as apposed an ant-viral effect, it is most likely less prone to the development of resistance. Larger studies would have needed to be undertaken to determine if the administration of emetine to uncomplicated cases reduced the likelihood of severe disease. Other noteworthy observations raised by Points:My mortality with the emetine series of cases was virtually nil, for the two cases that did die were beyond hope when the drug was given. …… One had already been sick a week, and her family persistently refused to let me use this treatment until the night before her death; and the other had been sick five days before I got hold of her, and was already well advanced in pneumonia when given the first dose. Even then the beneficial effects of the drug could be seen, the patient’s temperature dropped, and they seemed better for a while, but the delirium and coma steadily grew worse and they passed off.“Each case treated with emetine received injections, given from twelve to twenty-four hours apart, according as my time would permit my seeing the patient once or twice a day.Unfortunately I did not use the emetine treatment sufficiently early in the epidemic to determine its true value. The disease was on the wane when I began to use it, so whether or not it would produce the same marvelous results in all cases as it did in these is a question.This problem now presents itself to my mind: Is emetine a cure for the complicated cases of Influenza?”50 value of 0.16 µM. The mode of action (MOA) of emetine, as an antiviral agent, has not been fully elucidated. However, the MOA may depend on the virus in question. For the coronaviruses, emetine interferes with the viruses’ ability to infect a cell. Shen et al., 2019 performed viral infectivity studies and determined that emetine was an entry inhibitor that blocked MERS-CoV infection almost completely . compare50 in the low-nM range (~0.04 µM), partly indicating that the antiviral effect is most likely post-entry and at the step of viral replication [50 of 0.121 µM [On the other hand, Yang et al., 2018, in order to understand the stage of the Zika virus life cycle affected by emetine, varied compound treatment before, during, and after Zika virus inoculation . For alllication . In furt0.121 µM . As a potential other MOA, Mukhopadhyay et al., 2016, for the human cytomegalovirus (HCMV), found that inhibition by emetine depended on 40S ribosomal processing S14 (RPS14) binding to MDM2. Emetine lead to a disruption of the HCMV-induced MDM2-p53 interaction. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment confirming a new mode of action .Tang et al., 2020 demonstrated, by methods described previously  that emeBoth Tang et al., 2020  demonstrIn the treatment of herpes zoster infections emetine has demonstrated very consistent, rapid, persistent and effective pain relief and wound healing, as demonstrated by numerous authors, at very low doses—in the 2−7 centigrain (~1.2−3.6 mg) dosage range with many patients only requiring 1 or 2 doses and most given under five doses on alternate days ,53,54,56p ≤ 0.05) relative to control. Emetine also significantly reduced  the swelling induced by hyaluronidase relative to control. The formalin inflammation experiment was monitored for changes in induced inflammation at multiple time points during the first seven hours and the following 10 days. From this last experiment the anti-inflammatory effects increased and peaked in the seventh hour, closely matching the latency seen in patients treated for shingles [Before the antiviral properties of emetine were known, Jorda and Rothschild 1958  set out shingles . Furthershingles . Anothershingles .interferon” or the like, in vitro, or in vivo studies to explain a clinical observation. Suffice to say that it is well known that emetine inhibits protein synthesis and this action is understood to be responsible for the amoebicidal action of emetine [Several studies looked at the action of emetine on interferon/s and various aspects of the immune system ,69,70,71 emetine ,73,74. ITo further support this argument it was shown that the human mTOR   transcriThe toxicity emetine will be described in terms of the toxicity likely to be seen in clinical practice and the toxicity seen in the laboratory.The toxicity of emetine, like most drugs, is dose-related. With a long biological half-life it can accumulate, and that accumulation does increase the risk of toxicity. This toxicity of emetine will be explored in terms of low and high doses of emetine. Low doses will be defined as doses less than 6 mg (with many studies only using ~3 mg) of emetine daily. The majority of studies employ doses of less than 6 mg per day. High doses will be defined as between ~20 mg and 60 mg. When emetine was used widely, the increased risk of emetine toxicity in the elderly/frail was very much recognized and usually warranted a reduction of the amoebiasis dose by at least half (~30 mg or less). In some situations, emetine would have been contraindicated but at times there was little choice, especially with regards to treating liver abscesses.The information below is not intended to be a replacement for appropriate prescribing information or other drug monographs.Low dose: No study referenced in this paper using low dose emetine highlighted a cardiac concern.High dose: Cardiac toxicity has traditionally been the largest concern of emetine therapy. Yang et al. 1980 reviewed the cardiovascular side effects of therapeutic doses of emetine, concluding that cardiac toxicity frequently included ECG changes and hypotension (discussed below) and occasionally tachycardia and precordial pain. These changes occur during treatment or after completion of treatment and often last a period of time. The patient usually recovers without any change in cardiovascular function [function .ECG changes, particularly flattening or inversion of the T-wave and prolongation of the Q-T interval, occur in many (25−50%) patients ,79. HoweLow dose: the only study that mentioned blood pressure at all was Griveaud and Achard 1959 who described the hypotensive effect as minimal and of a short duration (“not durable”) but believed it was important to monitor [ monitor .High dose: It is said that the hypotension from emetine is rarely marked [Occasional patients exhibited a fall in blood pressure, rarely more than 15 or 20 mm. of mercury, which usually occurred between the sixth and ninth doses” [y marked . Howevery marked . Klatskiy marked . In a prh doses” . Unlike h doses” .Low dose: Very few of the studies using low-dose emetine reported any nausea. Most noted that there was no nausea or did not comment on it at all. Griveaud, reporting on 5 years’ experience with emetine, noted that a few of the patients had some nausea [e nausea . One stue nausea .High dose: The incidence of nausea in patients taking ameobiasis doses of emetine are highly variable. Heilig [not a single case suffered from nausea, vomiting or toxic diarrhea, from neuritis or muscular weakness” and suggested that “it is possible that these favorable results are partly due to the absence of toxic contaminations (cephaeline) in the brand of emetine that is used in our hospital”. On the other hand, Klatskin 1948 [Nausea occurred in almost a third of the subjects and was only occasionally accompanied by vomiting. In many it appeared within two hours of an injection and subsided rapidly, but in others it was more persistent”. Interestingly, this study used several commercial preparations of emetine hydrochloride [. Heilig  commentekin 1948 , with 10chloride .3 antagonists, such as ondansetron [4 receptor with little activity on 5HT3 [Nausea from the ipecac alkaloids is relatively easy to treat. The emetic effects of ipecac syrup (30 mL) can be completely eliminated, and nausea significantly reduced, by the use of 5HTansetron . Emetine on 5HT3 . HoweverPain on injection is included because it is a common feature of both subcutaneous and intramuscular injection and for low and high doses of emetine. In relation to intramuscular injections (high dose); pain can be almost immediately after injection and disappear in a few hours, but in the others the pain may not appear until a day or two later. There can be local tenderness, poorly localized, constant aching of the injected muscle, or more commonly, of all the surrounding muscles. The aching and tenderness usually lasts for several days to a week after treatment is stopped. Emetine is rarely discontinued because of pain .Traditionally, the preferred routes of emetine administration are by deep subcutaneous or intramuscular injection. The intravenous use of emetine was considered contraindicated because it was thought to be too dangerous and offered no therapeutic advantage . HoweverParmer and Cottrill 1949 observed that soon after injection of emetine in rabbits, the heart levels were relatively high, but after 6 h had dropped off to the low levels seen over the next 6 days . To reduFor low-dose emetine a 6 mg dose could be given, subcutaneously or intramuscularly, in divided doses to minimize risk.50 and, where determined, half-maximal cytotoxic concentration (CC50) values and the selectivity index  calculated. The CC50 values show a large degree of variation depending on the type of CC50 test, cell line and the duration of drug exposure.50 values of 8 µM and less, and comparing those values to the conservative tissue concentrations achieved in 50.There is a large disparity between the toxicity seen in the laboratory to what is seen in practice. For example, looking at the number of studies in 50 tests use a method that indirectly measures metabolic activity  or determines cell proliferation (cell counting). It is assumed that a decrease in metabolic activity or a proliferation is an indicator of cell toxicity or death. This is not always the case. Emetine is a protein synthesis inhibitor known to inhibit the 40S subunit of a ribosome [There are several reasons that could account for these discrepancies. Several CCribosome ,89. Thisribosome .50 tests, in relation to emetine, is the cell lines used to determine toxicity. Emetine is well known to exhibit anti-cancer properties [50 are immortal cell lines, arguably sharing several characteristics of cancer. Their sensitivity to emetine could depend on how the cells were immortalized . Toxicity seen in these cell lines may not be reflective of what happens to non-cancerous cell lines in vivo. This action is also seen in non-cancer cell lines. Emetine was shown to inhibit hyper-proliferating pulmonary artery smooth muscle cells (PASMCs) from rats with pulmonary hypertension, normalizing proliferation/apoptosis. However, emetine had minimal effect on non-hyper-proliferating PASMCs. This action was hypothesized to be due in part to the normalization of hypoxia-inducible factors under hypoxic conditions [Another limitation of CCoperties ,92. The nditions .50 and SI data, in the case of emetine as an antiviral, is of little value. Emetine has been used extensively in millions of humans over many years for the treatment of amoebiasis and the more relevant in vivo toxicity is well described and understood. Furthermore, as the antiviral and potentially anti-inflammatory doses being suggested are substantially lower than that used for the treatment of amoebiasis any toxicity will be significantly lower.Overall, the value of in vitro CC50 data  for SARS-CoV2. At this low dose, the side effects associated with the normal treatment dose of amoebiasis are unlikely to be a clinical problem. Traditional contraindications of kidney or heart disease are also much less likely to be an issue. Based on ECThe rapid reversal of cyanosis seen with Spanish influenza appeared to be an effect independent of emetine’s antiviral activity. If anything, the Spanish influenza virus was arguably resistant to emetine, with no decrease in temperature or a reduction of any symptoms or shortening of the length of the disease in mild cases. Added to that a patient’s cholecystitis pain being relieved by emetine further suggests an anti-inflammatory response, given that emetine has no analgesic properties . There iAt what dose are you likely to see an anti-inflammatory effect? In this article, we hypothesized that the anti-inflammatory MOA is the same as the antiviral and anti-amoebic MOA—through the inhibition of protein synthesis—in particular, binding to the human 40S subunit of the ribosome. If this is correct then the anti-inflammatory doses may be very similar to the antiviral doses (low). However, the effect seen by Points  for the Is a loading dose necessary? Loading doses were never used for the treatment of amoebiasis. It would not have been possible to safely administer such large doses. For antiviral doses a single large dose could be a loading dose or, because of its long biological half-life, a sustained-release dose. However, it is unknown if RNA needs to be detached from the 40S subunit in order for emetine to attach. If this is the case multiple doses may have a better outcome.What is the likely duration of therapy? Emetine protein synthesis inhibition is considered irreversible ,98. TherWhat alternatives are there to emetine? A potential alternative to emetine is the very structurally similar, and completely synthetic dehydroemetine. An injection form of dehydroemetine is still available in India from Tablets India Limited, under the brand name Tilemetin. It is currently unknown if this agent has similar antiviral and anti-inflammatory properties, and it should be tested if emetine is found to be effective. Dehydroemetine is believed to be safer agent than emetine in terms of cardiac toxicity . Glaxo, Can emetine be used in combinations with other drugs to improve antiviral efficacy or reduce side effects? The concept of antiviral synergism is well known and understood. With regards to emetine, Choy et al., 2020 demonstrated that emetine and remdesivir, when used in combination, displayed significant antiviral synergism [Cephaëlis acuminata or Cephaëlis ipecacuanha from which emetine can be obtained. While these plants are commercially grown, similar wild species/varieties are endangered [If emetine proves to be successful for the treatment of SARS-CoV2, either as an antiviral or an anti-inflammatory agent, then this would place a huge stress on wild populations of dangered ,13. Therdangered ,103,104.Emetine, shows promising antiviral and anti-inflammatory properties. The use of low-dose, or even, high-dose emetine should be considered in the treatment of COVID-19. Other viruses are far more sensitive to the effects of emetine than SARS-CoV2, for which emetine may prove useful.


Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming “RNAP.mRNA.ribosome” complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate. Due to the scarcity of intracellular membrane-delimited compartmentalization, prokaryotic cells have historically been regarded as spatially unorganized. The lack of a nuclear membrane that physically separates the chromosomal DNA from the cytosolic environment led to the well-accepted notion that transcription and translation are spatiotemporally coupled in bacteria and archaea. Coupled transcription-translation (CTT) occurs when ribosomes bind and start to translate nascent mRNAs, whose transcription has not terminated yet, therefore forming an “RNAP·nascent mRNA·ribosome” complex .in vitro, an active force exerted by translating ribosomes could be necessary to release the mRNAs from their templates. Indirectly, he implied that transcription and translation could be spatiotemporally coupled . It was assumed for years that, in the absence of ribosomes engaged in CTT, Rho utilization (rut) sites in the nascent transcripts become exposed and are readily recognized by Rho, which translocates towards RNAP and causes the disassembly of the transcription elongation complex . This state favors Rho recognition of the rut sites in the nascent mRNA, which is followed by the closure of the Rho ring and disassembly of the TEC ppGpp has recently emerged as an important player in CTT coordination. Upon amino acid starvation, the ribosome-associated RelA detects uncharged tRNAs at the A-site of the ribosomes and triggers the rapid synthesis of (p)ppGpp , inducinRecent publications have expanded the (p)ppGpp targetome , and addAlarmones also coordinate transcription and translation less straightforwardly. It has recently been shown that (p)ppGpp binds inosine-guanosine kinase, which prevents the excessive accumulation of purine nucleotides and leads to pRpp synthesis, a precursor molecule for pyrimidine nucleotides, tryptophan, and histidine . Thus, avia (p)ppGpp ensures that RNAP elongation rates decrease accordingly and maintains CTT coordination without the need for physical contacts of the transcription and translation machineries. Considering the ongoing discovery of (p)ppGpp-regulated processes and the tight relationship of alarmones with the metabolic status of the cell, we expect that additional pathways ensuring CTT coordination via (p)ppGpp will be discovered. These complementary mechanisms, i.e., physical contacts between RNAP and the leading ribosome and alarmone-mediated coordination, account for the observed correlation between transcription and translation elongation rates under different growth conditions.The evidence presented above supports a scenario where CTT coordination is preserved by different, although complementary means, depending on the growth conditions . When trE. coli, although their conclusions were not always in agreement ppGpp (see section Coordination of CTT). In other cases, this kinetic coupling precedes the formation of a coupled expressome. Specifically, RNAP tends to pause within the first 100 nt after the start codon  ratio , such asly mixed . This fa. Indeed, several lines of evidence have recently challenged the classical view that transcription is inherently coupled to translation, and the global occurrence of CTT has been questioned.Coupled transcription-translation is widely accepted by the microbiology community and supported by extensive work. Yet, most knowledge regarding CTT emanates either from M. pneumoniae (The Fredrick lab developed a hammerhead ribozyme-based reporter system that enables measuring and comparing protein synthesis carried out by limited vs. unlimited number of translation rounds . They apeumoniae . This inSimilar conclusions emanated from studying the relationship between termination efficiency (TE) at intrinsic terminators and their intragenic position . TE of tWhereas these studies argue against the idea of physical CTT, they are not incompatible with the existence of RNAP-ribosome complexes remotely connected by nascent mRNAs. Yet, further evidence supports the idea that transcription and translation in some cases are completely uncoupled. It is accepted that translation increases mRNA stability by ribosome shielding against ribonucleolytic attack . A reassE. coli cells . Although Rho is recruited to TECs early after transcription initiation , this “runaway” transcription creates extensive distance between RNAP and the leading ribosome, supporting the idea that transcription and translation are mostly uncoupled in B. subtilis. Accordingly, transcription terminators in B. subtilis are located only a few nucleotides downstream of stop codons  Prevent association between the leading ribosome and the nascent mRNA, once the SD sequence emerges from RNAP. (2) Protect the ribosome-free transcript from ribonucleolytic decay in the cytoplasm. (3) Carry out translation-uncoupled transcription without disruption by intrinsic and Rho-dependent terminators.E. coli, RNAP shows pronounced transcriptional pausing at sites overlapping with SD sequences and start codons  belong to an evolutionarily widespread family of small, acidic proteins that were originally discovered as mediators of cold shock response, but it is currently understood that their function exceeds adaptation to cold  perform extraribosomal moonlighting functions as free RBPs, often involved in gene regulation  or through an alternative mechanism remains unknown. Additionally, S1 was shown to increase protein secretion that is mediated by the hemolysin signal peptide, and this was accompanied by the stabilization of the corresponding transcripts  operon, which is subjected to an additional layer of regulation by L4 that is degradosome-independent. Specifically, L4 overexpression increases the stability of the tnaCAB transcript but causes translational repression of the TnaA protein by binding to the spacer between tnaC-tnaA , Hfq exerts post-transcriptional regulation in  in cirA  and mutStS mRNAs , as welltS mRNAs . In all  of RBSs , which ced genes .Salmonella , CsrA represses translation of the virulence effector NleA  sites decreased Rho-dependent termination at specific cases where nut and rut sites overlap  mechanisms. Opposite to dedicated antiterminators, PA factors associate with and modify TECs to promote transcriptional readthrough over multiple transcription terminators distally located in the operons under their regulation . For exaochaetes . Deletiogulation . All in cspA gene takes place at all temperatures, but the cspA transcript is highly unstable at 37°C due to RNase E-mediated decay  systems  interact with RNAP and facilitate Rho-dependent termination  and a rut site that is necessary for Rho-mediated transcription termination of this operon  sequence, located in the intergenic region between the second and third genes of the operon ppGpp-mediated CTT regulation shows great potential. Alarmone-deficient strains are metabolically compromised but viable, so, unlike compounds that target essential targets, targeting (p)ppGpp metabolism may greatly reduce cell viability without exerting the selective pressure that leads to the arousal of antibiotic resistance .E. coli  can offer hints of how to purposely disrupt CTT under conditions that it is essential. Most likely, these mechanisms act in cooperation. In some cases, simultaneous action of UTT-promoting factors may be required, as suggested by the existence of RNA-mediated Hfq-CsrA complexes in E. coli  and the ruginosa . Likewisotection . RNPs thotection , could pe operon . Similar RNase E , and csped by S1 . Besidesm RNases . Thus, RChlamydomonas, where they also bind and localize mRNAs to specific sites where local translation takes place (By now, the notion that prokaryotes have intricate intracellular organization is well-acknowledged . Conseques place . Hence, In the past, researchers have drawn a clear line between transcriptional and post-transcriptional regulatory mechanisms in bacteria. In the light of the emerging evidence, we posit that many post-transcriptional regulatory mechanisms come into action on nascent transcripts and that their cotranscriptionality has so far been ignored. As already recognized in eukaryotes , factorsMIO and OAC wrote the original draft and edited the manuscript. OAC coordinated the work and acquired funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genomes of polyomaviruses are characterized by their tripartite organization with an early region, a late region and a noncoding control region (NCCR). The early region encodes proteins involved in replication and transcription of the viral genome, while expression of the late region generates the capsid proteins. Transcription regulatory sequences for expression of the early and late genes, as well as the origin of replication are encompassed in the NCCR. Cell tropism of polyomaviruses not only depends on the appropriate receptors on the host cell, but cell-specific expression of the viral genes is also governed by the NCCR. Thus far, 15 polyomaviruses have been isolated from humans, though it remains to be established whether all of them are genuine human polyomaviruses (HPyVs). The sequences of the NCCR of these HPyVs show high genetic variability and have been best studied in the human polyomaviruses BK and JC. Rearranged NCCRs in BKPyV and JCPyV, the first HPyVs to be discovered approximately 30 years ago, have been associated with the pathogenic properties of these viruses in nephropathy and progressive multifocal leukoencephalopathy, respectively. Since 2007, thirteen novel PyVs have been isolated from humans: KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, STLPyV, HPyV12, NJPyV, LIPyV and QPyV. This review describes all NCCR variants of the new HPyVs that have been reported in the literature and discusses the possible consequences of NCCR diversity in terms of promoter strength, putative transcription factor binding sites and possible association with diseases. Polyomaviruses (PyVs) are non-enveloped viruses that are typically 40–45 nm in diameter, and that possess a double-stranded circular genome of around 5000 base-pairs. Birds and mammals, including humans, are natural hosts for PyVs ,2. RecenWhether all of these are genuine human polyomaviruses (HPyVs) remains to be determined. BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV6, HPyV, TSPyV, HPyV9, HPyV10, STLPyV, HPyV12, and NJPyV are classified as human polyomaviruses by the International Committee of Taxonomy of Viruses ,20, LIPyFunctionally, the PyV genome is tripartite consisting of the early region, the late region, and the noncoding control region (NCCR) A. The eaStudies with simian virus 40 (SV40 or Macaca mulatta polyomavirus 1) and murine polyomaviruses have been pivotal in unveiling the functions of this region. The SV40 NCCR contains the origin of replication (ori), which consists of GRGGC motifs to which LT binds and is flanked by an AT-rich sequence and an easily denaturated imperfect palindrome ,36. BindThe NCCR of the HPyVs varies between 267 bp  to 645 bp  see B. This rThe diversification based on the presence of a certain NCCR rearranged structure contributed to determining HPyVs strains as “archetype” or prototype”. The importance of the NCCR rearrangements during HPyVs infection became obvious when different strains of JCPyV were examined. The archetype JCPyV NCCR strain (CY) is divided into six boxes named A (36 bp), B (23 bp), C (55 bp), D 66 bp), E (18 bp), and F (69 bp) and contains the origin of replication (ORI), the promoter and the enhancer elements . The NCC bp, E , which includes the origin of replication and a TATA-box, P (68 bp), Q (39 bp), R (63 bp), and S (63 bp), containing TATA-like elements and the regulatory regions for early and late genes expression, is considered the infectious strain, shed in the urine of immunocompetent individuals ,70,71. ASimilarly to JCPyV, the plausible instability of the archetype BKPyV NCRR could contribute to the development of the prototype strains, which is able to cause polyomavirus-associated nephropathy in kidney transplant recipients and hemorrhagic cystitis in hematopoietic stem cell transplant recipients ,79,80,81Additional NCCR structures have been described for both viruses ,86,87,88LT/sT gene and the start codon of the VP2 gene, of the novel HPyVs and their known effect on promoter activity. We discuss how NCCR rearrangements may affect the binding of putative transcription factors, and whether specific NCCR configurations are associated with disease.Little is known about the genetic diversity of the NCCRs from the novel HPyVs and the biological relevance in terms of viral transcription, replication, and possible pathogenic properties. In this review, we provide an overview of the mutations in the NCCR, which is defined as the sequence between the start codon of the KIPyV has been mostly isolated from oral and respiratory specimens from (pediatric) patients with respiratory diseases that suffer from other viral and bacterial infections (reviewed in ). WhetheMost KIPyV NCCR sequences are obtained from nasopharyngeal swabs or aspirates, but also from blood form healthy blood donors  and fromThe KIPyV NCCR contains putative binding sites for several transcription factors . The effA total of 185 partial or complete WUPyV NCCR sequences are available in the GenBank . All strNCCR rearrangements are described as a pivotal event in the onset of HPyVs-related pathology, as demonstrated for JCPyV and BKPyV, in which NCCRs not only control gene expression, but also serve as the main determinants in viral replication, containing the origin of DNA replication and transcription factor binding sites ,105. MCPWhether a MCPyV variant with a particular NCCR architecture is associated with specific patient groups is not known. MCPyV with different NCCRs have been characterized in Merkel cell carcinoma samples see , while fThe relative early and late promoter strength of seven MCPyV NCCR variants was compared in human dermal fibroblasts, and in the non-classical MCPyV-positive MCC cell line MCC13 . All varAlthough HPyV6 and HPyV7 DNA is commonly present in the normal skin of healthy persons ,123, HPySeventeen HPyV6 NCCR sequences are deposited in GenBank. Four of them are sequences obtained from HPyV6 DNA amplified in sewage , six are from healthy skin , two are from bile samples (Bile-72 and Bile-81), and two are combined nose and throat samples from kidney transplant patients (QLD-49Br and QLD-61Br). One sample was obtained from pruritic skin lesion (UTSW6.1), one from a lymph node from a patient with an angiolymphoid hyperplasia with Kimura disease (LN1), and one from a nasopharyngeal aspirate of a child with respiratory tract infections (BJ376) see . Two cliSequences of 15 different HPyV7 NCCR isolates are available in GenBank . Six are from healthy skin specimens, while nine are clinical samples from patients, including skin lesions from lung or renal transplant patients with rash and pruritus, and skin lesions from patients with pruritic and dyskeratotic dermatoses . The mutThe early promoter activity of the PITT1 and PITT2 variants was significantly higher than the activity of the reference strain in the colon adenocarcinoma cell line SW480, whereas a tendency to lower activity in human embryonal kidney HEK293 cells was observed . The proInterestingly, the NCCR of the recently isolated QPyV DNA shows >80% identity with the HPyV7 NCCR , while tTwenty-four TSPyV NCCR sequences are deposited in the GenBank . Most saHPyV9 was originally detected in the serum and urine from a renal transplant patient under immunosuppressive treatment . ShortlyTwenty NCCR sequences are available in GenBank , with thSequences of 7 STLPyV NCCRs are available in GenBank . These vThe NCCR sequence of two HPyV12 isolates is known. One of them carries a 26 bp deletion in the distal part of the late promoter (nucleotides 297–322) (Only one human and one feline LIPyV isolate have been reported, and they differ by four-point mutations . No NJPySimilar to BKPyV and JCPyV, novel HPyV isolates with mutations in their NCCR are commonly detected in human samples. However, for the most recently described PyVs isolated from human specimens, none or very few isolates have been reported, and large deletions and/or duplication are lacking. Our knowledge of the effect of mutations in the NCCR on viral promoter activity and viral replication is incomplete because only a few studies have addressed the effect of NCCR mutations on the promoter activity, while the impact on viral replication has not been examined. Replication studies have been hampered by the lack of permissive cell systems for all novel HPyVs, except dermal fibroblasts which support productive MCPyV infection . The HPy


In silico analysis is a promising approach for understanding biological events in complex diseases. Herein we report on the innovative computational workflow allowed to highlight new direct interactions between human transcription factors (TFs) and an entire genome of virus ZikaSPH2015 strain in order to identify the occurrence of specific motifs on a genomic Zika Virus sequence that is able to bind and, therefore, sequester host’s TFs. The analysis pipeline was performed using different bioinformatics tools available online (free of charge). According to obtained results of this in silico analysis, it is possible to hypothesize that these TFs binding motifs might be able to explain the complex and heterogeneous phenotype presentation in Zika-virus-affected fetuses/newborns, as well as the less severe condition in adults. Moreover, the proposed in silico protocol identified thirty-three different TFs identical to the distribution of TFBSs (Transcription Factor Binding Sites) on ZikaSPH2015 strain, potentially able to influence genes and pathways with biological functions confirming that this approach could find potential answers on disease pathogenesis. The Zika virus (ZIKV) is a positive-sense, single-strand RNA arbovirus belonging to the Flaviviridae family, genus Flavivirus, which includes other well known viruses, such as yellow fever, the dengue virus and the West Nile virus . In 1947The infection causes a disease known as “Zika virus disease“, which is asymptomatic or paucisintomatic in approximately 80% of cases, and when symptomatic, adult populations experience low-grade fever, often accompanied by malaise, headache, dizziness, anorexia, conjunctivitis with retro-orbital pain, arthritis, transient arthralgia and maculopapular rash, which appear between 3 and 12 days after infection , scientiZIKV is spread by numerous Aedes species including Aedes aegypti, Aedes albopictus and Aedes henselli. In North America and Europe, Aedes albopictus is spreading as a new vector of ZIKV (spread from Gabon), as previously reported. The genus Aedes mosquito is common in warm climates; in the northern hemisphere it is present in North America and in different areas of the Mediterranean. Before 2007, a few cases of infections were reported at different places in Asian and African countries. Changes in the last years, due to human activities and climate changes, likely induced adaptive evolution mutation in the RNA genome of ZIKV . FurtherEpidemic spread and the finding of autochthonous cases of ZIKV infection in various countries caused an international public health emergency. The health organizations therefore focused great attention on the potential risk of ZIKV transmission by transfusion of labile blood components  . AdditioConsidering the impact of the ZIKV infection and its clinical consequences, studies are warranted to understand reasons underlining differences in clinical presentation, particularly with regards to occurrence of fetal abnormalities. The ZIKV genome is a single-stranded of ~10.500 nt with an RNA 5′ cap structure that codes one polyprotein that contains three structural peptides (capsid protein (C), envelope (E) and membrane (prM/M) glycoproteins) and seven nonstructural proteins  ..15].http://meme-suite.org/tools/meme-chip) [The entire ZikaSPH2015 strain was analyzed using a MEME-ChIP that performs a comprehensive motif analysis (including motif discovery) on large sets of sequences identified by ChIP-seq or CLIP-seq experiments on Human DNA (me-chip) .http://meme-suite.org/doc/tomtom.html), another tool of MEME suite that compared the motifs against a database of known motifs . Hocomoco is a complete collection of transcription factor binding models for humans, using a large-scale ChIP-Seq analysis. Tomtom ranked the motifs in the database and produced an alignment for each significant match, searching one or more query motifs against one or more databases of human target motifs (and their reverse complements when applicable). The report for each query was a list of target motifs, ranked by p-value in the order that the queries appear in the input file. The E-value and the q-value for each match were also reported. The q-value is the minimal false discovery rate at which the observed similarity would be considered significant. Tomtom estimated q-values from all the match p-values using the Benjamini and Hochberg method. By default, significance was measured by q-value of the match [All motifs identified were used as query for Tomtom , another software tool, to identify the most related correlation within query TFs set using a guilt-by-association approach. The bioinformatics tool used a large database of functional interaction net-works from multiple organisms, and each related TF is traceable to the source network used to make the prediction [The list of TFs was loaded in STITCH  on ZikaSPH2015 strain is shown in the Using STITCH analysis, twenty-three of those TFs were connected in a gene regulatory knowledge network, while ten seemed to be out of this network and had no interactions .This in silico approach allowed us to predict recruitment of some TFs directly related to different clinical manifestation (some of them syndromic) characterized by growth retardation, dysmorphic features, intellectual disability and others.In particular, the analysis revealed a possible reduction of BCL11A  TFs reported in association with intellectual developmental disorder with persistence of fetal hemoglobin (HbF) including microcephaly, down-slanting palpebral fissures, strabismus and external ear abnormalities .The decrease of POU1F1  is implicated in growth retardation combined with pituitary hormone deficiency , reporteIndications regarding the relationship of PRDM6  and ZFP28  were put in evidence by our approach. PRDM6 is associated with isolated nonsyndromic patent ductus arteriosus, while ZFP28 plays an essential role in controlling gene expression during cardiac and vascular pathogeneses ,23. ThisMoreover, our analysis highlighted DUX4 , which, when depleted, is related to muscle weakness or atrophy, one of the main characteristics of Guillaume-Barrett syndrome . Indeed,Another group of TFs, highlighted by this approach, were those involved in immune processes as they relate to immune specific regulatory elements. In particular, ZNF680 (Zinc Finger Protein 680), IRF4 , SPI1  and MEIS1 , which are involved in differentiation and activation of macrophages or B-cells ,30. In tNFE2L2 , ZNF250 (Zinc Finger Protein 250) and ZNF549 (Zinc Finger Protein 549) were activated in response to infection  and inflammation. The sequestration of these TFs makes the cells more susceptible to the virus infection ,36.By this approach, the indications on possible recruitment of different TFs involved in embryonic development was also underlined. In detail, the following have been indicated: LYL1  , SNAI1  is associated with early-onset of insulin dependent diabetes mellitus ; NKX6-1 However, the analysis performed did not revealed a direct correlation with microcephaly.Based on the performed in silico analysis, it was possible to identify the occurrence of specific motifs in the ZikaSPH2015 strain. These motifs could be able to bind specific host (human) TFs, and, consequently, recruitment/subtraction could be provoked. In this manner, as we hypothesized, a haploinsufficiency-like effect is occurring, with consequences on involved target genes which are predicted by our in silico approach. Even though many studies are concentrated on the molecular mechanism of ZIKV infection, the exact mechanisms that induce the complex symptomatology, including microcephaly and brain anomalies in fetuses, remain unclear. An intricate network of TFs has been suggested by our analysis . InteresThe Zika virus remains ambiguous in its role in development of severe microcephaly . In fact, specific TFs directly related to microcephaly were not evidenced by our approach. Our data is suggesting, according to recent observations, that microcephaly could be a result of the combined insufficiency of several embryonic neurological developmental TFs, able to produce a synergic effect on target genes which provoke this condition ; or, othIn conclusion, the suggested pipeline is easy to access, and this easily reproducible approach could be used not only to understand the mechanism of infection and therefore the characterization of TFs involved, but also to identify metabolic networks suitable to identify tissue specific biomarkers and possible pathway dysfunction, which are useful for development of a possible vaccine or therapeutic strategy . The application of these integrated analysis pipelines, which can be developed in a single user-friendly interface, could help support infection investigations by providing rapid information on multiple datasets analysis , especially in times of crisis such as those currently in progress with SARS-CoV-2 .Certainly, some of the differences observed in the phenotypic responses may be caused by interindividual genetic variations, but the identification of these regulatory elements should increase diagnostic sensitivity and accuracy of cohort classification, and therefore guide treatment .