Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.     

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31, 46, 57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5′ type 1 cap and conserved RNA structures at both the 5′ and 3′ untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. ​(Fig.1).1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, NS3, and NS5 are cytoplasmic. NS2A, NS2B, NS4A, and NS4B are largely hydrophobic transmembrane proteins with small hydrophilic segments (Fig. ​(Fig.1).1). The post- and cotranslational cleavage of the polyprotein is performed by NS3 in the cytoplasm and by host proteases in the ER lumen to yield the mature proteins (Fig. ​(Fig.1)1) (33, 43). Of the nonstructural proteins, NS3 and NS5 are the best characterized, and both are essential for viral replication (23, 27, 41). Both proteins are multifunctional. The N-terminal one-third of NS3 contains the viral protease (NS3pro), which requires a portion of NS2B for its activity, while the remaining portion codes for the RNA helicase/NTPase/RTPase domain (NS3hel) (21, 22, 32, 55). NS5, however, contains both an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (16, 51). The functions of NS1, NS2A, NS4A, and NS4B are not well understood, but they appear to play important roles in replication and virus assembly/maturation and have been found to bind to NS3 and NS5, possibly modulating their activity (33, 36).Open in a separate windowFIG. 1.Schematic diagram of flavivirus polyprotein organization and processing. (Top) Linear organization of the structural and nonstructural proteins within the polyprotein. (Middle) Putative membrane topology of the polyprotein predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (indicated by arrows). (Bottom) Different complexes that are thought to arise in different cellular compartments during and following polyprotein processing.Because of its enzymatic activities and its critical role in viral replication and polyprotein processing, NS3 constitutes a promising drug target for antiviral therapy (31). NS3pro (residues 1 to 169) is a trypsin-like serine protease with the characteristic catalytic triad (Asp-His-Ser) and a highly specific substrate recognition sequence, conserved in all flaviviruses, consisting of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1′ (11). NS3pro has an aberrant fold compared to the canonical trypsin structure, and its folding and protease activity are dependent on a noncovalent association with a central 47-amino-acid hydrophilic domain of NS2B (19, 21). The remainder of NS2B contains three transmembrane helices involved in membrane associations. NS3 mediates cleavages at the C-terminal side of the highly conserved dibasic residue located at the coding junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 and also between the C terminus of C and NS4A (11, 33) (Fig. ​(Fig.11).The C-terminal portion of NS3 (NS3hel, residues 170 to 619) performs several catalytically related activities, namely, RNA strand separation and (poly)nucleotide hydrolysis (5, 22, 32, 55) at a common, RecA-like NTPase catalytic center that couples the energy released from the hydrolysis of the triphosphate moieties of nucleotides to RNA unwinding. Although the precise role of NS3 in replication has not been established, its helicase activity is thought to separate nascent RNA strands from the template strands and to assist replication initiation by unwinding RNA secondary structure in the 3′ untranslated region (11, 13, 15, 33). NS3 is a member of the DEAH/D box family within helicase superfamily 2 (SF2) and is characterized by seven conserved sequence motifs involved in nucleic acid binding and hydrolysis (45). In addition, its RNA triphosphatase activity is thought to be involved in the capping of the viral RNA. In the process of replication, NS3 interacts, most likely via its C-terminal domain, with NS5 (13, 15, 24, 26, 58, 62). The NS3 5′ triphosphatase and NS5 methyltransferase activities probably cooperate in cap formation by removing the terminal γ-phosphate and performing sequential N7 and 2′ O methylations, respectively (16, 28, 46, 56). The guanylyltransferase activity required for cap formation remains elusive at present, although recent evidence suggests that it may be present in NS5 (8, 17). In addition, the interaction between NS3 and NS5 can stimulate NS3 helicase/NTPase activity (15, 62).The atomic structures of NS3pro in the presence and absence of ligands and/or the NS2B activating domain (2, 19, 47) and NS3hel (35, 38, 39, 49, 58-60) are known, and recently, the structure of full-length DENV4 (one of four dengue virus serotypes) NS3 fused to an 18-residue NS2B cofactor (NS2B₁₈NS3) was reported (34). This structure revealed an elongated conformation, with the protease domain interfacing with the NTP binding pocket and being separated from NS3hel by a relatively flexible linker, which suggested that the protease domain may have a positive effect on the activity of the NTPase/helicase domain. However, other reports suggested that NS3pro has no or a very limited effect on the activity of NS3hel (32, 62). In addition, since current evidence suggests that NS2B is not part of the replication complex (Fig. ​(Fig.1)1) (36), and it is known that in the absence of the NS2B cofactor, NS3pro is unfolded and inactive, it becomes hard to envisage what effect the NS3 protease domain may have on the helicase domain in a biologically relevant context. Equally, it is still not clear what role the helicase domain plays during polyprotein processing by NS3pro and, in general, why these two apparently distinct and unrelated catalytic activities are harbored within a single polypeptide.In order to gain further insights into these questions, we report the biochemical analysis and crystallographic structure at a 2.75-Å resolution of full-length NS3 from Murray Valley encephalitis virus (MVEV), a member of the JEV group of flaviviruses, fused to the entire protease activation peptide of the NS2B cofactor (NS2B₄₅NS3). The structure reveals the protease and helicase domains to be structurally independent and differs dramatically from the structure observed for DENV4 NS2B₁₈NS3. We discuss the implications of this unexpectedly different configuration of the NS3 protein and argue that the structural flexibility observed is likely to be crucial for its multifunctional nature.     

Correction: Disease candidate gene identification and prioritization using protein interaction networks

Background  

Although most of the current disease candidate gene identification and prioritization methods depend on functional annotations, the coverage of the gene functional annotations is a limiting factor. In the current study, we describe a candidate gene prioritization method that is entirely based on protein-protein interaction network (PPIN) analyses.     

Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

Background  

Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.     

Inhibition of cysteine peptidase activity in ascitic fluid in pancreatic cancer patients

The work's objective is to answer the question whether there is any possibility of activity inhibition of cysteine peptidases inhibitors playing an important role in key processes accompanying cancer formation, including pancreas. There is a justified speculation that specific inhibitors of these enzymes may inhibit development of cancer processes by inhibiting their activity. In vitro studies confirmed that these enzymes in ascitic fluid were inhibited with egg whites inhibitors even to 90% of their original activity.     

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N<Superscript>pro</Superscript>) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N^pro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N^pro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N^pro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N^pro rabbit serum. When rabbits were immunized with the N^pro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N^pro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N^pro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N^pro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.     

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3-4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography-mass spectrometry (LC-MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE-SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE-SDS assay. The amount of the pre-LC peak recovered from the CE-SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb.     

Urinary Volatile Compounds as Biomarkers for Lung Cancer: A Proof of Principle Study Using Odor Signatures in Mouse Models of Lung Cancer

A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Although data supports the hypothesis that volatile compounds can be detected in the breath of lung cancer patients by the sense of smell or through bioanalytical techniques, analysis of breath samples is cumbersome and technically challenging, thus limiting its applicability. The hypothesis explored here is that variations in small molecular weight volatile organic compounds (“odorants”) in urine could be used as biomarkers for lung cancer. To demonstrate the presence and chemical structures of volatile biomarkers, we studied mouse olfactory-guided behavior and metabolomics of volatile constituents of urine. Sensor mice could be trained to discriminate between odors of mice with and without experimental tumors demonstrating that volatile odorants are sufficient to identify tumor-bearing mice. Consistent with this result, chemical analyses of urinary volatiles demonstrated that the amounts of several compounds were dramatically different between tumor and control mice. Using principal component analysis and supervised machine-learning, we accurately discriminated between tumor and control groups, a result that was cross validated with novel test groups. Although there were shared differences between experimental and control animals in the two tumor models, we also found chemical differences between these models, demonstrating tumor-based specificity. The success of these studies provides a novel proof-of-principle demonstration of lung tumor diagnosis through urinary volatile odorants. This work should provide an impetus for similar searches for volatile diagnostic biomarkers in the urine of human lung cancer patients.     

New species of Dialeurodes Cockerell (Hemiptera: Aleyrodidae) with diagnoses and keys to puparia and adults from Taiwan

Adult and immature stages of a new whitefly species Dialeurodes swidi Ko are described and illustrated. It can be distinguished from other species by the abundant large tubercles scattered over its dorsum, each of them bearing a geminate pore. The biology, comparative notes and identification keys to puparia and adults of Dialeurodes species of Taiwan are provided.