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Glucagon-like peptide-1 (GLP-1) is easily degraded by dipeptidyl peptidase-4 (DPP-4) in the human body, limiting its therapeutic effect on type II diabetes. Therefore, improving GLP-1 receptor agonist (GLP-1RA) stability is a major obstacle for drug development. We analyzed human GLP-1, DPP-4, and GLP-1 receptor structures and designed three GLP-1RAs, which were introduced into fusion protein fragments and changed in the overall conformation. This modification effectively prevented GLP-1RAs from entering the DPP-4 active center without affecting GLP-1RAs' ability to bind to GLP-1R, the new GLP-1RA hypoglycemic effect lasting for >24 h. Through molecular modeling, molecular dynamics calculation, and simulation, possible tertiary structure models of GLP-1RAs were obtained; molecular docking with DPP-4 and GLP-1R showed access to the fusion protein. The overall conformational change of GLP-1RAs prevented DPP-4 binding, without affecting GLP-1RAs' affinity to GLP-1R. This study provides important drug design ideas for GLP-1RA development and a new example for application of structural biology-based protein design in drug development. [ABSTRACT FROM AUTHOR]

To understand the origin of the spin-triplet Andreev reflection which may appear at a ferromagnet (F)/s-wave superconductor (S) interface, we study tunneling conductance spectra of F/2DEG/s-wave S tunnel junctions, with 2DEG the two-dimensional electron gas with Rashba spin-orbit coupling and subject to a magnetic field. It is found that both spin-flip and broken time-reversal symmetry in the 2DEG are conditions necessary to giving rise to the novel Andreev reflection and spin-triplet pairing correlations. [ABSTRACT FROM AUTHOR]

In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5′ half and a large, suboptimal intron in its 3′s half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (<5%) of ORF57 RNA undergoes double splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS 83636 . The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein. [ABSTRACT FROM AUTHOR]

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5′ splice site and two alternative 3′ splice sites, which produce E6&ast;I and E6&ast;II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6&ast;I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector''s exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5′ cap, with an optimal distance of less than 307 nt in order to facilitate better association of U1 small nuclear RNA with the intron 5′ splice site. The same was true for splicing of human β-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5′ cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression. [Copyright &y& Elsevier]

In this paper, we discuss the weighted Poincare´ inequalities on one-dimensional unbounded domains and give sufficient conditions for them to hold. [Copyright &y& Elsevier]

Human papillomaviruses (HPV) are a group of small non-enveloped DNA viruses whose infection causes benign tumors or cancers. HPV16 and HPV18, the two most common high-risk HPVs, are responsible for ~70% of all HPV-related cervical cancers and head and neck cancers. The expression of the HPV genome is highly dependent on cell differentiation and is strictly regulated at the transcriptional and post-transcriptional levels. Both HPV early and late transcripts differentially expressed in the infected cells are intron-containing bicistronic or polycistronic RNAs bearing more than one open reading frame (ORF), because of usage of alternative viral promoters and two alternative viral RNA polyadenylation signals. Papillomaviruses proficiently engage alternative RNA splicing to express individual ORFs from the bicistronic or polycistronic RNA transcripts. In this review, we discuss the genome structures and the updated transcription maps of HPV16 and HPV18, and the latest research advances in understanding RNA cis-elements, intron branch point sequences, and RNA-binding proteins in the regulation of viral RNA processing. Moreover, we briefly discuss the epigenetic modifications, including DNA methylation and possible APOBEC-mediated genome editing in HPV infections and carcinogenesis. [ABSTRACT FROM AUTHOR]

Presents information on a study which demonstrates that pyrimidine-rich exonic splicing suppressor (ESS) inhibits utilization of upstream three inch splice sites by blocking early steps in spliceosome assembly in bovine papillomavirus type 1. Materials and methods; Results of the study; Discussion.

In the publication of this article [1], there are a few errors in the article. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) from back-splicing have been found in every cell and tissue. By binding to miRNAs and proteins or even by encoding small proteins, circRNAs are now emerging as important regulators in modulating transcription, RNA splicing and interference. The highlighted discovery reports an important role of circRNAs in antiviral innate immunity by binding PKR as PKR inhibitors. Rapid degradation of circRNAs by activated RNase L from virus infection or poly I:C stimulation is required to free PKR for its activation. Systemic lupus erythematosus patients remark with circRNA reduction and aberrant PKR activation. [ABSTRACT FROM AUTHOR]

The corrected Figure 1a below shows the HPV16 P SB L sb promoter in the E7 open reading frame. Figure Graph: Figure 1 Papillomavirus genome structure, annotated ORFs, and conserved ACS in the origin of replication (Ori). In the published review [[1]], the HPV16 late promoter P SB L sb in Figure 1a was wrongly labeled in the E6 open reading frame. [Extracted from the article]

Approximately 5% of all human cancers are attributable to human papillomavirus (HPV) infections. HPV-associated diseases and cancers remain a substantial public health and economic burden worldwide despite the availability of prophylactic HPV vaccines. Current diagnosis and treatments for HPV-associated diseases and cancers are predominantly based on cell/tissue morphological examination and/or testing for the presence of high-risk HPV types. There is a lack of robust targets/markers to improve the accuracy of diagnosis and treatments. Several naturally occurring animal papillomavirus models have been established as surrogates to study HPV pathogenesis. Among them, the Cottontail rabbit papillomavirus (CRPV) model has become known as the gold standard. This model has played a pivotal role in the successful development of vaccines now available to prevent HPV infections. Over the past eighty years, the CRPV model has been widely applied to study HPV carcinogenesis. Taking advantage of a large panel of functional mutant CRPV genomes with distinct, reproducible, and predictable phenotypes, we have gained a deeper understanding of viral–host interaction during tumor progression. In recent years, the application of genome-wide RNA-seq analysis to the CRPV model has allowed us to learn and validate changes that parallel those reported in HPV-associated cancers. In addition, we have established a selection of gene-modified rabbit lines to facilitate mechanistic studies and the development of novel therapeutic strategies. In the current review, we summarize some significant findings that have advanced our understanding of HPV pathogenesis and highlight the implication of the development of novel gene-modified rabbits to future mechanistic studies. [ABSTRACT FROM AUTHOR]

RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection. Author summary: Like other herpesviruses, the expression of KSHV genes is in a tightly regulated manner during viral infection. The contribution of RNA splicing in the regulation of KSHV gene expression was extensively investigated in this report. We found that approximately one-third of all viral genes express spliced transcripts of which many undergo RNA alternative splicing. We identified a subset of viral RNA splicing events susceptible to the regulation of viral RNA-binding protein ORF57. This led to the discovery of ORF57-mediated regulation of the bicistronic ORF70-K3 RNA splicing and production of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3. Such regulation allows timely but mutually exclusive expression of these two viral proteins to execute their unique roles in immune evasion and viral genome replication during the KSHV lytic cycle. Thus, these data provide new insights into KSHV biology. Finally, our study represents a blueprint for RNA splicing studies of other pathogens in infected cells. [ABSTRACT FROM AUTHOR]

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰- 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors. Author summary: Persistent high-risk HPV infection leads viral DNA integration into the host genome and promotes viral carcinogenesis. We have been using the MmuPV1 mouse-infection model to study papillomavirus tumorigenesis and asked whether MmuPV1 DNA also integrates into the genomes of infected mouse cells. Strikingly, we found that MmuPV1 integration into the infected host genome, like high-risk HPV infections, is very common and the mapped integration sites were distributed on all of the mouse chromosomes. Consistently, we identified microhomology sequences in the range of 2–10 nts always at the integration junction regions. We further verified the MMEJ-mediated viral DNA integration in tumor tissues during MmuPV1 infection and a step-wise increase in the expression of the DNA repair MMEJ host factors from normal tissues, to tumor-free MmuPV1 infected tissues, and then to MmuPV1 tumors. Our observations provide the first evidence of MmuPV1 integration in virus-infected cells and a conceptual advance of how papillomavirus DNA integration contributes to the development of papillomavirus-associated precancers to cancers. [ABSTRACT FROM AUTHOR]

Abstract: Transport of mRNA from the nucleus to the cytoplasm is mediated by cellular RNA export factors. In this report, we examined how RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, function in modulating KSHV ORF57 expression. We found that knockdown of each factor by RNAi led to decreased ORF57 expression. Specifically, reduced expression of either UAP56 or RBM15 led to nuclear export deficiency of ORF57 RNA. In the context of the KSHV genome, the near absence of UAP56 or RBM15 reduced the expression of both ORF57 and ORF59 (an RNA target of ORF57), but not ORF50. Collectively, our data indicate that the expression of KSHV ORF57 is regulated by cellular RNA export factors and cofactors at the posttranscriptional level. [ABSTRACT FROM AUTHOR]

Abstract: Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection. [Copyright &y& Elsevier]

The weighted Poincaré inequalities in weighted Sobolev spaces are discussed, and the necessary and sufficient conditions for them to hold are given. That is, the Poincaré inequalities hold if, and only if, the ball measure of non-compactness of the natural embedding of weighted Sobolev spaces is less than 1. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA–RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA–RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors. [ABSTRACT FROM AUTHOR]

High-risk human papillomaviruses (HPVs) cause 5% of human cancers. Despite the availability of HPV vaccines, there remains a strong urgency to find ways to treat persistent HPV infections, as current HPV vaccines are not therapeutic for individuals already infected. We used a mouse papillomavirus infection model to characterize virus-host interactions. We found that mouse papillomavirus (MmuPV1) suppresses host immune responses via overexpression of stress keratins. In mice deficient for stress keratin K17 (K17KO), we observed rapid regression of papillomas dependent on T cells. Cellular genes involved in immune response were differentially expressed in the papillomas arising on the K17KO mice correlating with increased numbers of infiltrating CD8+ T cells and upregulation of IFNγ-related genes, including CXCL9 and CXCL10, prior to complete regression. Blocking the receptor for CXCL9/CXCL10 prevented early regression. Our data provide a novel mechanism by which papillomavirus-infected cells evade host immunity and defines new therapeutic targets for treating persistent papillomavirus infections. Author summary: Persistent infection with high-risk human papillomavirus may lead to a variety of cancers in human including cervical cancer, anal cancer, head and neck cancer and skin cancer. There has been a lack of animal model to study how papillomavirus infection evades cellular immune surveillance to establish long-term infections to cause cancers. We utilized a mouse papillomavirus, MmuPV1, as an infection model in immunocompetent FVB/N mice to study how the virus escapes immune recognition to establish persistent infections. We found a novel regulator, stress keratin 17 (K17), was induced in expression following MmuPV1 infection, and that K17 was critical for preventing T cell infiltration in MmuPV1-induced papillomas by inhibiting the CXCL9/CXCL10/CXCR3 axis. Our study provides a new mechanism by which papillomaviruses evade T cell response following infection. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies. Author summary: Molecular imaging diagnosis and targeted therapy have been successfully used for several types of tumors, but not yet applied to diagnose or treat EBV-associated NPC. Affibody molecules are small proteins engineered to bind to a large number of target proteins with high affinity, and therefore, can be developed as potential biopharmaceutical drugs for molecular diagnosis and therapeutic applications. In the present study, we screened and characterized EBV LMP-2-specific affibodies and evaluated their usage in molecular imaging of LMP-2 expressing cells and EBV LMP-2 tumor-bearing mice. Subsequently, we engineered and obtained an EBV LMP-2 affitoxin based on EBV LMP-2-binding affibodies and demonstrated its targeted cytotoxicity for EBV+ cell lines in vitro and in vivo. Our data indicate that the EBV LMP-2-specific affibody and its derived affitoxin are useful for diagnosis of LMP-2 expressing cells and targeted therapy of EBV-derived, LMP-2+ malignancies. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 Å and monomer at 3.0 Å. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167–222 of ORF57-CTD protrudes a long “arm” and holds the globular domains of the neighboring monomer, while the C-terminal residues 445–454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the “arm” region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the “arm” region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases. [ABSTRACT FROM AUTHOR]

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology. [ABSTRACT FROM AUTHOR]

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi’s sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection. [ABSTRACT FROM AUTHOR]

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains. [ABSTRACT FROM AUTHOR]

Summary: Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others. [ABSTRACT FROM AUTHOR]

RNA polyadenylation (pA) is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV) infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ∼90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site) in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3′RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A) signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6), K9 (vIRF1), K10.5 (vIRF3), K11 (vIRF2), K12 (Kaposin A), T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression. [ABSTRACT FROM AUTHOR]

Background: Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes. Methods: Genomic DNA sequences of 8 high- risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real- time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates. Results: A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215- 221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established. Conclusions: This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner. [ABSTRACT FROM AUTHOR]

Network resilience is vital for the survival of networks, and scale-free networks are fragile when confronted with targeted attacks. We survey network robustness to targeted attacks from the viewpoint of network clients by designing a unique mechanism based on the undeniable roles of network clients in real-world networks. Especially, the mechanism here is designed on the actual phenomenon that the vital nodes in a network may be totally different for clients with different demands. Concretely, node client-demand centrality is proposed to quantify the contributions of nodes to network clients and we show that it is a proper index to assign an order to network nodes according to node importance for network clients. Great discrepancy of node importance order for clients with different demands is found in scale-free networks with four different kinds of link weight distribution, which suggests that the destructiveness of fatal attacks on networks can be greatly reduced by adjusting the demands of network clients. [ABSTRACT FROM AUTHOR]

Abstract: We identified a novel inhibitor of growth family member 2 (ING2) isoform, ING2b, which shares exon 2 with ING2a, but lacks the N-terminal p53 binding region. Contrary to ING2a, ING2b’s promoter has no p53 binding sites. Consistently, activation of p53 led to suppression of ING2a, leaving ING2b unaffected. Through isoform-specific targeting, we showed that ING2a knockdown suppressed cell growth only in the presence of p53, ING2b knockdown had no effect on cell growth, and knockdown of both induced cell cycle arrest and apoptosis independently of p53. ING2a and ING2b have compensatory roles that protect cells from cell cycle arrest and apoptosis and may be involved in development of chemotherapeutic resistance. [Copyright &y& Elsevier]

Summary: In lower eukaryotes, Sir2 serves as a histone deacetylase and is implicated in chromatin silencing, longevity, and genome stability. Here we mutated the Sirt1 gene, a homolog of yeast Sir2, in mice to study its function. We show that a majority of SIRT1 null embryos die between E9.5 and E14.5, displaying altered histone modification, impaired DNA damage response, and reduced ability to repair DNA damage. We demonstrate that Sirt1+/−;p53+/− mice develop tumors in multiple tissues, whereas activation of SIRT1 by resveratrol treatment reduces tumorigenesis. Finally, we show that many human cancers exhibit reduced levels of SIRT1 compared to normal controls. Thus, SIRT1 may act as a tumor suppressor through its role in DNA damage response and genome integrity. [Copyright &y& Elsevier]

We have read with great interest the Correspondence by Shibo Jiang and colleagues,[1] in which they propose a name change for the newly emerged coronavirus,[2] which was recently designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses.[3] The authors argued that the use of SARS in the virus name could confuse the public about the disease that it causes; in addition, they noted that the name SARS-CoV-2 is not consistent with the disease name chosen by WHO, coronavirus disease 2019. The SARS-CoV species includes viruses such as SARS-CoV, SARS-CoV PC4-227, and SARSr-CoV-btKY72. The use of SARS in naming SARS-CoV-2 does not derive from the name of the SARS disease but is a natural extension of the taxonomic practice for viruses in the SARS species. [Extracted from the article]

Abstract: A kinetic model of ubiquinone-10 production by batch fermentation with Agrobacterium tumefaciens ATCC4452 was firstly studied. After observation of experimental data, a model based on the logistic equation of ATCC4452 growth, ubiquinone-10 accumulation combined non-growth-associated and growth-associated contributions, and consumption of sugar for biomass formation and the maintenance of biomass, was developed. The optimal set of parameters was estimated by fitting the model to experimental data. The results predicted by the model were in good agreement with the experimental observations. Based on estimated kinetic parameters and characteristics of product formation and substrate utilization, a strategy of fed-batch operation was adopted in order to obtain more ubiquinone-10 accumulation. The final production of ubiquinone-10 reached 71.5mgl−1 in 96h of incubation, corresponding to more 3.5 times than that of batch operation, in fed-batch fermentation using four stage feeding strategy. [Copyright &y& Elsevier]

Anal squamous cell carcinoma is the most frequent virus-related non-AIDS-defining neoplasia among HIV-infected individuals, especially MSM. The objectives of this study were to analyze the effectiveness of the quadrivalent HPV (qHPV) vaccine to prevent anal ≥ high-grade squamous intraepithelial lesions (≥HSILs), external ano-genital lesions (EAGLs), and infection by qHPV vaccine genotypes in HIV+ MSM, and to study the immunogenicity of the vaccine and risk factors for ≥ HSILs. This study is nested within a randomized, double-blind, placebo-controlled trial of the qHPV vaccine, which enrolled participants between May 2012 and May 2014, with a 48-month follow-up. A vaccine or placebo was administered at 0, 2, and 6 months, and vaccine antibody titers were evaluated at 7, 12, 24, 36, and 48 months. Data were gathered at 12, 24, 36, and 48 months on sexual habits, CD4/CD8 cell/counts, HIV viral load, and the results of cytology (Thin Prep® Pap Test), HPV PCR genotyping (Linear Array HPV Genotyping Test), and high-resolution anoscopy (Zeiss 150 fc© colposcope). The study included 129 patients (mean age of 38.8 years, 40 [31%] with a history of AIDS, 119 [92.2%] receiving ART, and 4 [3.3%] with virological failure), 66 (51.2%) in vaccine arm and 63 (48.4%) in placebo arm. The vaccine and placebo groups did not differ in ≥ HSILs (14.1 vs. 13.1%, respectively, p = 0.98) or EAGL (11.1 vs. 6.8%, p = 0.4) rates during follow-up; however, a protective effect against HPV 6 was observed during the first year of follow-up in the vaccine versus placebo group (7.5% vs. 23.4%; p = 0.047). A between-arm difference (p = 0.0001) in antibodies against qHPV vaccine genotypes was observed at 7 months (76.9% in vaccine arm vs. 30.2% in placebo arm), 12 months (68.1% vs. 26.5%), 24 months (75% vs. 32.5%), 36 months (90% vs. 24.4%), and 48 months (87.2% vs. 30%). Finally, the factor associated with the risk of anal ≥ HSIL onset during the four-year follow-up was the receipt of the last dose of the vaccine less than 6 months earlier in comparison to those vaccinated for a longer period (82.4% vs. 17.6% (OR 0.869 [95% CI, 0.825–0.917]). Vaccine and placebo arms did not significantly differ in ≥ HSIL or EAGL rates or in protection against infection by HPV genotype vaccine except for HPV6 at 12 months after the first dose. A long-lasting immune response was observed in almost all the vaccinated men. The main protective factor against ≥ HSIL was to have completed the vaccination regimen more than 6 months earlier. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 regulates viral gene expression at the posttranscriptional level during viral lytic infection. To study its function in the context of the viral genome, we disrupted KSHV ORF57 in the KSHV genome by transposon-based mutagenesis. The insertion of the transposon into the ORF57 exon 2 region also interrupted the 3′ untranslated region of KSHV ORF56, which overlaps with the ORF57 coding region. The disrupted viral genome, Bac36-Δ57, did not express ORF57, ORF59, K8α, K8.1, or a higher level of polyadenylated nuclear RNA after butyrate induction and could not be induced to produce infectious viruses in the presence of valproic acid, a histone deacetylase inhibitor and a novel KSHV lytic cycle inducer. The ectopic expression of ORF57 partially complemented the replication deficiency of the disrupted KSHV genome and the expression of the lytic gene ORF59. The induced production of infectious virus particles from the disrupted KSHV genome was also substantially restored by the simultaneous expression of both ORF57 and ORF56; complementation by ORF57 alone only partially restored the production of virus, and expression of ORF56 alone showed no effect. Altogether, our data indicate that in the context of the viral genome, KSHV ORF57 is essential for ORF59, K8α, and K8.1 expression and infectious virus production. [ABSTRACT FROM AUTHOR]