Complex of the herpes simplex virus type 1 origin binding protein UL9 with DNA as a platform for the design of a new type of antiviral drugs

The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80?bp). The protein also binds to a single-stranded DNA (OriS^?) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3′-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5′- and 3′- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А?+?Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.     

Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM

In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the stop codons with different affinities and trigger the hydrolysis of the ester bond that links the polypeptide with the P-site tRNA. Cryo-electron microscopy (cryo-EM) results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center. These results are similar to those obtained for RF2, but there is an important difference in how the factors bind to protein L11, which forms part of the GTPase-associated center of the large ribosomal subunit. The difference in the binding position, C-terminal domain for RF2 versus N-terminal domain for RF1, explains a body of L11 mutation studies that revealed differential effects on the activity of the two factors. Very recent data obtained with small-angle X-ray scattering now reveal that the solution structure of RF1 is open, as here seen on the ribosome by cryo-EM, and not closed, as seen in the crystal.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Age as a determinant for dissemination of seasonal and pandemic influenza: an open cohort study of influenza outbreaks in Östergötland County, Sweden

An understanding of the occurrence and comparative timing of influenza infections in different age groups is important for developing community response and disease control measures. This study uses data from a Scandinavian county (population 427.000) to investigate whether age was a determinant for being diagnosed with influenza 2005-2010 and to examine if age was associated with case timing during outbreaks. Aggregated demographic data were collected from Statistics Sweden, while influenza case data were collected from a county-wide electronic health record system. A logistic regression analysis was used to explore whether case risk was associated with age and outbreak. An analysis of variance was used to explore whether day for diagnosis was also associated to age and outbreak. The clinical case data were validated against case data from microbiological laboratories during one control year. The proportion of cases from the age groups 10-19 (p<0.001) and 20-29 years old (p<0.01) were found to be larger during the A pH1N1 outbreak in 2009 than during the seasonal outbreaks. An interaction between age and outbreak was observed (p<0.001) indicating a difference in age effects between circulating virus types; this interaction persisted for seasonal outbreaks only (p<0.001). The outbreaks also differed regarding when the age groups received their diagnosis (p<0.001). A post-hoc analysis showed a tendency for the young age groups, in particular the group 10-19 year olds, led outbreaks with influenza type A H1 circulating, while A H3N2 outbreaks displayed little variations in timing. The validation analysis showed a strong correlation (r = 0.625;p<0.001) between the recorded numbers of clinically and microbiologically defined influenza cases. Our findings demonstrate the complexity of age effects underlying the emergence of local influenza outbreaks. Disentangling these effects on the causal pathways will require an integrated information infrastructure for data collection and repeated studies of well-defined communities.     

Effect of DNA Local Conformation on the Affinity and Binding Specificity of bis-Netropsins to DNA

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked tail-to-tail via cis-diammineplatinum group (Nt–Pt(NH ₃ )–Nt) or aliphatic pentamethylene chain (Nt–(CH ₂ ) ₅ –Nt), has been studied. Both bis-netropsins have been shown to bind to DNA oligomer 5"-CCTATATCC-3" (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5"-CCXCC-3" [where X = TTATT (II), TTAT (III), TTTTT (IV), and AATTT (V)] along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of Nt–(CH ₂ ) ₅ –Nt with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin ( ₂) to monodentate binding constant ( ₁) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for Nt–Pt(NH ₃ )–Nt and Nt–(CH ₂ ) ₅ –Nt binding as a hairpin has been found to be DNA duplex 5"-CGTATACG-3".     

Associations between adult spectral tarsiers

While anecdotal observations of gregarious behavior in nocturnal prosimian primates are common, most anthropologists continue to refer to them as solitary, perhaps based on the assumption that the occasional social interactions observed via ad libitum methods represent random chance encounters and not patterned social interactions. In this paper, I test the null hypothesis that nocturnal encounters between spectral tarsier (Tarsius spectrum) group members, outside of the sleeping tree, are the result of chance. Three male‐female pairs were radio‐collared and observed over a 4‐month period, using continuous focal animal sampling at the Tangkoko Nature Reserve (Sulawesi, Indonesia). Using Waser's random gas model, I found that spectral tarsiers spent more time in proximity to other group members than expected by chance, given the size of their home range and nightly path length. Adult group members spent 11% of the night in physical contact and an additional 17% of the night within a 10‐m radius of one another. Spectral tarsiers were also observed to significantly increase the amount of time spent foraging when located less than 10 m from another group member. Individuals foraging in proximity to another adult group member had lower insect capture rates compared to individuals who were not foraging in proximity to another adult group member. If living in a group is costly to these tarsiers' foraging efficiency, then why don't they actively avoid one another when foraging? One situation in which it might benefit tarsiers to be gregarious is high predation pressure. Preliminary results suggest that predation pressure by snakes may be the most likely factor selecting for the tarsiers to forage in proximity. Am J Phys Anthropol 128:74‐83, 2005. © 2005 Wiley‐Liss, Inc.