Evaluating serum level of thymidylate synthase in post burn keloid patients before and after intralesional injection of 5-fluorouracil

Background Keloids are fibrous lesions formed at the site of trauma due to types I and III collagen irregular production. The presence of thymidylate synthase (TS) is a must for DNA synthesis and repairs causing cell death. 5-fluorouracil (5-FU) is a fluorinated pyrimidine analogue acting as an anti-metabolic agent that inhibits thymidylate synthase and interferes with ribo-nucleic acid (RNA) synthesis. Objectives we aimed to evaluate the level of thymidylate synthase in post burn keloid patients before and after intralesional injection of 5-fluorouracil. Methods The study included 20 keloid patients and 20 healthy subjects as a control. Serum TS was estimated using commercially available enzyme-linked immunosorbent assay (ELISA) kits before and after treatment with 5-fluorouracil. Results There was a statistically significant difference in TS levels before and after 5-FU treatment (p < 0.05). Also, results have shown that 5-FU injection has good satisfactory results in treatment of keloid causing reduction in scar volume and symptoms improvement (90% of the patients improved). On the other hand, there was no statistically significant difference in TS levels and the outcomes of the treatment. Conclusion Our findings suggest that intralesional 5-FU injection in keloid has very satisfactory results. However, thymidylate synthase enzyme has a minimal role in evaluating the treatment of keloid, so further studies are required to elaborate the relation between this enzyme and keloid scars.

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

Functional interaction between the RNA exosome and the sirtuin deacetylase Hst3 maintains transcriptional homeostasis

Eukaryotic cells maintain an optimal level of mRNAs through unknown mechanisms that balance RNA synthesis and degradation. We found that inactivation of the RNA exosome leads to global reduction of nascent mRNA transcripts, and that this defect is accentuated by loss of deposition of histone variant H2A.Z. We identify the mRNA for the sirtuin deacetylase Hst3 as a key target for the RNA exosome that mediates communication between RNA degradation and transcription machineries. These findings reveal how the RNA exosome and H2A.Z function together to control a deacetylase, ensuring proper levels of transcription in response to changes in RNA degradation.

Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis

Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order (Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.

Hepatitis delta virus genome RNA synthesis initiates at position 1646 with a non-templated guanosine

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense ssRNA virus that produces three RNAs in infected cells: genome, antigenome and mRNA; the latter encodes hepatitis delta antigen, the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5’ end of the genome, including the synthesis start site and its chemical composition, are not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling detection of the 5’ end of the genome RNA. The 5’ end of this RNA is capped, as expected for a Pol II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA Polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how synthesis of the viral RNA begins, nor even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed hairpin-like structure of the viral RNA. The 5’ end of the RNA is capped, as expected for Pol II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses Pol II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3’ and 5’ promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.