Requirement of a 5′-Proximal Linear Sequence on Minus Strands for Plus-Strand Synthesis of a Satellite RNA Associated with Turnip Crinkle Virus

AbstractViral RNA replication begins with specific recognition of cis-acting RNA elements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated host factors. A short RNA element (3′-AACCCCUGGGAGGC) located 41 bases from the 5′ end of minus strands of satellite RNA C (satC), a 356-base subviral RNA naturally associated with turnip crinkle virus (TCV), was previously identified as important for plus-strand synthesis using an in vitro RdRp assay (H. Guan, C. Song, A. E. Simon, 1997, RNA 3, 1401–1412). To examine the functional significance of this element in RNA replication, mutations were introduced into the consecutive C residues in the element. A single mutation of the 3′-most C residue resulted in undetectable levels of satC plus strands when transcripts were assayed in protoplasts and suppressed transcription directed by the element in vitro. However, satC minus strands were detectable at 6 h postinoculation (hpi) of protoplasts, accumulating to about 10% of wild-type levels at 24 hpi. This mutation, when in the plus-sense orientation, had little or no effect on minus-strand synthesis from full-length satC plus strands in vitro, suggesting that the 5′-proximal RNA element is required for satC plus-strand synthesis. In addition, in vivo genetic selection revealed a strict requirement for 10 of the 14 nucleotides of the element, indicating that the primary sequence is essential for RNA accumulation

NF-κB has a Direct Role in Inhibiting Bmp- and Wnt-Induced Matrix Protein Expression.

The host response to pathogens through nuclear factor κB (NF-κB) is an essential defense mechanism for eukaryotic organisms. NF-κB-mediated host responses inhibit bone and other connective tissue synthesis and are thought to affect the transcription of matrix proteins through multiple indirect pathways. We demonstrate that inhibiting NF-κB in osteoblasts increases osteocalcin expression in vivo in mice with periodontal disease. Mutating NF-κB binding sites on osteocalcin (OC) or bone sialoprotein (Bsp) promoters rescues the negative impact of NF-κB on their transcription and that NF-κB can inhibit Wnt- and Bmp-induced OC and Bsp transcription, even when protein synthesis is inhibited, indicating a direct effect of NF-κB. This inhibition depends on p65-p50 NF-κB heterodimer formation and deacetylation by HDAC1 but is not affected by the noncanonical NF-κB pathway. Moreover, NF-κB reduces Runx2 and β-catenin binding to OC/Bsp promoters independently of their nuclear localization. Thus, inflammatory signals stimulate the direct interaction of NF-κB with response elements to inhibit binding of β-catenin and Runx2 binding to nearby consensus sites and reduce expression of matrix proteins. This direct mechanism provides a new explanation for the rapid decrease in new bone formation after inflammation-related NF-κB activation

Herpes Simplex Virus-1 Infection in Human Primary Corneal Epithelial Cells is Blocked by a Stapled Peptide that Targets Processive DNA Synthesis

Purpose: Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus −1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. Methods: A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. Results: The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. Conclusions: Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis. © 202

Seizing the window of opportunity to mitigate the impact of climate change on the health of Chinese residents

The health threats posed by climate change in China are increasing rapidly. Each province faces different health risks. Without a timely and adequate response, climate change will impact lives and livelihoods at an accelerated rate and even prevent the achievement of the Healthy and Beautiful China initiatives. The 2021 China Report of the Lancet Countdown on Health and Climate Change is the first annual update of China’s Report of the Lancet Countdown. It comprehensively assesses the impact of climate change on the health of Chinese households and the measures China has taken. Invited by the Lancet committee, Tsinghua University led the writing of the report and cooperated with 25 relevant institutions in and outside of China. The report includes 25 indicators within five major areas (climate change impacts, exposures, and vulnerability; adaptation, planning, and resilience for health; mitigation actions and health co-benefits; economics and finance; and public and political engagement) and a policy brief. This 2021 China policy brief contains the most urgent and relevant indicators focusing on provincial data: The increasing health risks of climate change in China; mixed progress in responding to climate change. In 2020, the heatwave exposures per person in China increased by 4.51 d compared with the 1986–2005 average, resulting in an estimated 92% increase in heatwave-related deaths. The resulting economic cost of the estimated 14500 heatwave-related deaths in 2020 is US$176 million. Increased temperatures also caused a potential 31.5 billion h in lost work time in 2020, which is equivalent to 1.3% of the work hours of the total national workforce, with resulting economic losses estimated at 1.4% of China’s annual gross domestic product. For adaptation efforts, there has been steady progress in local adaptation planning and assessment in 2020, urban green space growth in 2020, and health emergency management in 2019. 12 of 30 provinces reported that they have completed, or were developing, provincial health adaptation plans. Urban green space, which is an important heat adaptation measure, has increased in 18 of 31 provinces in the past decade, and the capacity of China’s health emergency management increased in almost all provinces from 2018 to 2019. As a result of China’s persistent efforts to clean its energy structure and control air pollution, the premature deaths due to exposure to ambient particulate matter of 2.5 μm or less (PM2.5) and the resulting costs continue to decline. However, 98% of China’s cities still have annual average PM2.5 concentrations that are more than the WHO guideline standard of 10 μg/m3. It provides policymakers and the public with up-to-date information on China’s response to climate change and improvements in health outcomes and makes the following policy recommendations. (1) Promote systematic thinking in the related departments and strengthen multi-departmental cooperation. Sectors related to climate and development in China should incorporate health perspectives into their policymaking and actions, demonstrating WHO’s and President Xi Jinping’s so-called health-in-all-policies principle. (2) Include clear goals and timelines for climate-related health impact assessments and health adaptation plans at both the national and the regional levels in the National Climate Change Adaptation Strategy for 2035. (3) Strengthen China’s climate mitigation actions and ensure that health is included in China’s pathway to carbon neutrality. By promoting investments in zero-carbon technologies and reducing fossil fuel subsidies, the current rebounding trend in carbon emissions will be reversed and lead to a healthy, low-carbon future. (4) Increase awareness of the linkages between climate change and health at all levels. Health professionals, the academic community, and traditional and new media should raise the awareness of the public and policymakers on the important linkages between climate change and health.</p

Replication and 3\u27-end repair of a subviral RNA associated with turnip crinkle virus

Replication of plus (+)-strand RNA viruses proceeds through minus (−)-strand intermediates. Satellite RNA C (satC), one of the nonessential subviral RNAs of Turnip crinkle virus (TCV), is dependent on the TCV-encoded RdRp for its replication. Earlier work showed that a stem-loop structure at the 3′ end of (+)-strand satC is required for synthesis of (−)-strands (Song and Simon, 1995a). Using an in vitro RdRp assay, I defined two separate cis-acting elements on satC (−)-strands that can promote complementary strand synthesis. One element comprises 11 bases and is located near the 3′ end (3′-proximal), and the other consists of 14 bases and is located 41 bases from the 5′ end (5′ -proximal). Both elements contain multiple consecutive C residues followed by multiple consecutive purines. In vivo mutagenesis and genetic selection (SELEX) studies were carried out to investigate the functional significance of the two elements as well as the satC (−)-strand 3′ terminus (3 ′ OH-CCCUAU), which contains the (−)-strand 3′ -end sequence 3′ OH-CC1–2(A/U)(A/U)(A/U) found in all carmovirus RNAs (named the carmovirus consensus sequence or CCS). My results indicate that the 3′-terminal CCS and the 5′-proximal element are highly conserved and required for satC (+)-strand synthesis. Although mutations introduced into the 3′-proximal element were tolerable, this element preferentially contains a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to satC accumulation in vivo. All RNAs associated with TCV terminate with the motif CCUGCCC-3 ′ at the 3′ end. Transcripts of satC containing a deletion of the motif, or the 3′-terminal 6 bases, are nearly always repaired to wild-type in vivo by RdRp-mediated primer extension of oligoribonucleotides synthesized by abortive initiation and complementary to the 3′ end of TCV genomic RNA (Nagy et al., 1997). In this thesis, I provide evidence that two additional mechanisms are used by the TCV RdRp to repair shorter deletions of the 3 ′-end motif of satC. Deletion of the 3′-terminal CCC residues along with addition of 8 non-template bases is repaired in vivo mainly by homologous recombination between the similar 3 ′ ends of satC and TCV. Deletion of the 3′-terminal 4 or 5 bases, in the presence or absence of non-template bases, led to recovery of progeny containing a mixture of wild-type 3′ ends and non-wild-type 3′ ends that included base alterations, deletions and insertions. Assays using an in vitro RdRp transcription system indicate that the TCV RdRp is likely able to polymerize nucleotides in a template-independent, non-random fashion before initiating transcription of deletion-containing satC. The existence of 3 different repair mechanisms associated with a single virus suggests an intrinsic need for 3′ -end reconstruction in the cellular environment

Tumorigenic Adenovirus Type 12 E1A Inhibits Phosphorylation of NF-κB by PKAc, Causing Loss of DNA Binding and Transactivation▿

Human adenovirus type 12 (Ad12) E1A protein (E1A-12) is the key determinant of viral tumorigenesis. E1A-12 mediates major histocompatibility complex class I (MHC-I) shutoff by inhibiting the DNA binding of the transcriptional activator NF-κB (p50/p65) to the class I enhancer. This enables Ad12 tumorigenic cells to avoid class I recognition and lysis by cytotoxic T lymphocytes. In this study, we demonstrate that the phosphorylation of p50 and p65 by the catalytic subunit of protein kinase A (PKAc) is essential for NF-κB DNA binding and transactivation activity. Treatment with H89 and knockdown of PKAc in cells led to the inhibition of phosphorylation at p50 Ser337 and p65 Ser276 and loss of DNA binding by NF-κB. Importantly, NF-κB phosphorylation by PKAc was repressed by tumorigenic E1A-12, but not by nontumorigenic Ad5 E1A (E1A-5). The stable introduction of E1A-12 into Ad5 nontumorigenic cells resulted in a decrease in the phosphorylation of NF-κB, loss of NF-κB DNA binding, and the failure of NF-κB to activate a target promoter, as well as diminution of MHC-I transcription and cell surface expression. Significantly, the amount and enzymatic activity of PKAc were not altered in Ad12 tumorigenic cells relative to its amount and activity in nontumorigenic Ad5 cells. These results demonstrate that E1A-12 specifically prevents NF-κB from being phosphorylated by PKAc