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At least 487 records · Page 27
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors

ABSTRACT The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC 50 ]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC 50 ). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PA N target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro . Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs. IMPORTANCE The effectiveness of antiviral drugs can be severely compromised by the emergence of resistant viruses. Therefore, determination of the mechanisms by which viruses become resistant is critical for drug development and clinical use. RO-7 is a compound that potently inhibits influenza virus replication and belongs to a new class of drugs in late-stage clinical trials for treatment of influenza virus infection. Here we demonstrate that a single amino acid change acquired under prolonged virus exposure to RO-7 renders influenza viruses significantly less susceptible to its inhibitory effects. We have discovered how the mutation can simultaneously interfere with drug activity and still maintain efficient virus replication. These findings have important implications for the development of more effective derivatives of RO-7-like drugs and provide guidance for how to monitor the emergence of resistance.

Jones, Jeremy C.↗
DOE OSTI2024

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Say where you sample: Increasing site selection transparency in urban ecology

Urban ecological studies have the potential to extend our understanding of socio-ecological systems beyond that of an individual city or region. Cross-comparative empirical work and synthesis are imperative to develop a general urban ecological theory. This can be achieved only if studies are replicable and generalizable. Transparency in methods reporting facilitates generalizability and replicability by documenting the decisions scientists make during the various steps of research design; this is particularly true for sampling design and selection because of their impact on both internal and external validity and the potential to unintentionally introduce bias. Three interdependent aspects of sample design are study sample selection (e.g., specific organisms, soils, or water), sample specification (measurement of specific variable of interest), and site selection (locations sampled). Of these, documentation of site selection—the where component of sample design—is underrepresented in the urban ecology literature. Using a stratified random sample of 158 papers from 12 major urban ecology journals, we investigated how researchers selected study sites in urban ecosystems and evaluated whether their site selection methods were transparent. We extracted data from these papers using a 50-question, theory-based questionnaire and a multiple-reviewer approach. Our sample represented almost 45 years of urban ecology research across 40 different countries. We found that more than 80% of the papers read were not transparent in their site selection methodology. We do not believe site selection methods are replicable for 70% of the papers read. Key weaknesses include incomplete descriptions of populations and sampling frames, urban gradients, sample selection methods, and property access. Low transparency in reporting the where methodology limits urban ecologists’ ability to assess the internal and external validity of studies’ findings and to replicate published studies; it also limits the generalizability of existing studies. The challenges of low transparency are particularly relevant in urban ecology, a field where standard protocols for site selection and delineation are still being developed. These limitations interfere with the fields’ ability to build theory and inform policy. We conclude by offering a set of recommendations to increase transparency, replicability, and generalizability.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1. Genotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as those of genotype 1b and a strain of HCV that replicates with exceptional efficiency (JFH-1, classified as genotype 2a). Indeed, unlike the inefficient genotype 1b NS3, which has been well studied, JFH-1 NS3 is a superhelicase with strong RNA affinity and high unwinding efficiency on a broad range of targets. Crystallographic analysis reveals architectural features that promote enhanced biochemical activity of JFH-1 NS3. These findings show that even within a single family of viruses, drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Toward the fabrication of a 5- μm -resolution Wolter microscope for the National Ignition Facility (invited)

Advancements in computer-controlled polishing, metrology, and replication have led to an x-ray mirror fabrication process that is capable of producing high-resolution Wolter microscopes. Here, we present the fabrication and test of a nickel–cobalt replicated full-shell x-ray mirror that was electroformed from a finely figured and polished mandrel. This mandrel was designed for an 8-m source-to-detector-distance microscope, with 10× magnification, and was optimized to reduce shell distortions that occur within 20 mm of the shell ends. This, in combination with an improved replication tooling design and refined bath parameters informed by a detailed COMSOL Multiphysics® model, has led to reductions in replication errors in the mirrors. Mandrel surface fabrication was improved by implementing a computer-controlled polishing process that corrected the low-frequency mandrel figure error and achieved <2.0 nm RMS convergence error. X-ray tests performed on a pair of mirror shells replicated from the mandrel have demonstrated <10 μm full-width at half-maximum (FWHM) spatial resolution. Here, we discuss the development process, highlight results from metrology and x-ray testing, and define a path for achieving a program goal of 5 μm FWHM resolution.

47 OTHER INSTRUMENTATION↗
DOE OSTI2016

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Absence of clinical disease and contact transmission of HPAI H5NX clade 2.3.4.4 from North America in experimentally infected pigs

Background In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged. Objectives Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America. Methods Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT‐PCR, virus isolation, hemagglutination inhibition, and ELISA. Results Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT‐PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen‐specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross‐reactivity between wild‐type avian strains. All RRT‐PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs. Conclusions H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine.

H5↗
DOE OSTI2022

Nonclassical autophagy activation pathways are essential for production of infectious Influenza A virus in vitro

Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta-transcriptome of IAV and host gene expression dynamics during early (1–3 hpi), mid (4–6 hpi), and late (8–12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress-activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK-Thr187 signaling was crucial for the production of infectious viral particles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of recG genetic interactions in Escherichia coli by transposon sequencing

ABSTRACT Maintaining the integrity of the genome is of utmost importance for cell division and propagation. In Escherichia coli , the RecG protein has been implicated in processing branched recombination intermediates during DNA repair processes, but the primary cellular role(s) of RecG and the repair pathways in which it acts have been difficult to define. To gain additional insight into RecG function, we employed transposon sequencing to identify recG genetic interactions and reveal complementary or redundant functions. The strongest hits from the screen were the dam , uvrD , rnhA , radA , and rep genes. The conditional importance of these five genes in cells lacking recG was confirmed using a plasmid-based assay, revealing synthetic lethal interactions for most double deletion strains. Several of the synthetic lethal gene combinations (with uvrD, rep, and radA , but not rnhA or dam ) were suppressed by deletion of recF or recO , indicating that their genetic relationships involved roles in post-replication gap repair. Additionally, loss of the RecG/SSB interaction phenocopied a recG deletion when combined with dam , uvrD , radA , or rnhA deletions but not with rep . The results reinforce the idea of RecG as a general genome guardian. RecG has at least two functions. It plays an important role in the resolution of joint molecules behind the fork, formed during post-replication gap repair. RecG is also required to suppress genome over-replication caused by unscheduled replication initiation at R-loops, at double-strand breaks caused by dam inactivation, and during replication termination. IMPORTANCE DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalisgenerates a new host for the analysis of biomass deconstruction

Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria. To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host. The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.

09 BIOMASS FUELS↗
DOE OSTI2015

Transmitted virus fitness and host T cell responses collectively define divergent infection outcomes in two HIV-1 recipients

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro , while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

ASFV Gene A151R Is Involved in the Process of Virulence in Domestic Swine

African swine fever virus (ASFV) is the etiological agent of a swine pandemic affecting a large geographical area extending from Central Europe to Asia. The viral disease was also recently identified in the Dominican Republic and Haiti. ASFV is a structurally complex virus with a large dsDNA genome that encodes for more than 150 genes. Most of these genes have not been experimentally characterized. One of these genes, A151R, encodes for a nonstructural protein and has been reported to be required for the replication of a Vero-cell-adapted ASFV strain. Here, we evaluated the role of the A151R gene in the context of the highly virulent field isolate Georgia 2010 (ASFV-G) during virus replication in swine macrophage cell cultures and during experimental infection in swine. We show that the recombinant virus ASFV-G-ΔA151R, harboring a deletion of the A151R gene, replicated in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the A151R gene is not required for ASFV replication in swine macrophages. Interestingly, experimental infection of domestic pigs demonstrated that ASFV-G-ΔA151R had a decreased replication rate and produced a drastic reduction in virus virulence. Animals were intramuscularly inoculated with 102 HAD50 of ASFV-G-ΔA151R and compared with pigs receiving a similar dose of virulent ASFV-G. All ASFV-G-infected pigs developed an acute lethal form of the disease, while those inoculated with ASFV-G-ΔA151R remained healthy during the 28-day observational period, with the exception of only one showing a protracted, but fatal, form of the disease. All ASFV-G-ΔA151R surviving animals presented protracted viremias with lower virus titers than those detected in ASFV-G-infected animals. In addition, three out of the four animals surviving the infection with ASFV-G-ΔA151R were protected against the challenge with the virulent parental virus ASFV-G. This is the first report indicating that the ASFV A151R gene is involved in virus virulence in domestic swine, suggesting that its deletion may be used to increase the safety profile of currently experimental vaccines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Unbalanced Nested Random Effects Estimation of Variance Components

To investigate the contributing factors of variance in the measurement of an iodine 127 (127I) sample, we implement a nested random effects analysis of variance (ANOVA). Historically, the reported uncertainty on a measurement of 127I has been obtained by methods of forward uncertainty propagation because there is typically only one replicate of a given sample for which to estimate the uncertainty. When samples are processed there are several types of quality control (QC) standards analyzed along-side the unknown samples with two to five replicates for each. Assuming the variance observed in these replicate QC standards is representative of that of the unknown samples, we use these data in a nested random effects ANOVA to estimate the total uncertainty of a measurement. We demonstrate this approach with two sets of measurements from Idaho National Laboratory and compare the results with the forward uncertainty propagation approach. Variance component estimates for the coarsest level of the nesting structure were most imprecise because replicates were most limited at these levels. We find that the results largely agree between forward propagation and ANOVA, and the greatest contributors of variance are due to instrument variation and chemical processing, with human processing being among the smallest contributors. This analysis provides reassurance that the reported uncertainties using forward propagation are reasonable and the process is well controlled. We propose a future designed experiment to increase replicates at the coarsest level of the hierarchy to improve estimates of these variance components.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner

Highlights: • Cdc6 protein is a component of the pre-replicative complex required for chromosomal replication initiation. • Cdc6 localized to centrosomes of S and G2 phases in a cell cycle-dependent manner. • The centrosomal localization was governed by centrosomal localization signal sequences of Cdc6. • Deletions or substitution mutations on the centrosomal localization signal interfered with centrosomal localization of the Cdc6 proteins. - Abstract: The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomes during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311–366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311–366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Multiple roles of genome-attached bacteriophage terminal proteins

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Residual Viremia in Treated HIV+ Individuals

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. However, some residual virus remains, below the level of detection, in HIV-infected patients on ART. Furthermore, the source of this viremia is an area of debate: does it derive primarily from activation of infected cells in the latent reservoir, or from ongoing viral replication? Our observations seem to be contradictory: there is evidence of short term evolution, implying that there must be ongoing viral replication, and viral strains should thus evolve. The phylogenetic analyses, and rare emergent drug resistance, suggest no long-term viral evolution, implying that virus derived from activated latent cells must dominate. We use simple deterministic and stochastic models to gain insight into residual viremia dynamics in HIV-infected patients. Our modeling relies on two underlying assumptions for patients on suppressive ART: that latent cell activation drives viral dynamics and that the reproductive ratio of treated infection is less than 1. Nonetheless, the contribution of viral replication to residual viremia in patients on ART may be non-negligible. However, even if the portion of viremia attributable to viral replication is significant, our model predicts (1) that latent reservoir re-seeding remains negligible, and (2) some short-term viral evolution is permitted, but long-term evolution can still be limited: stochastic analysis of our model shows that de novo emergence of drug resistance is rare. Thus, our simple models reconcile the seemingly contradictory observations on residual viremia and, with relatively few parameters, recapitulates HIV viral dynamics observed in patients on suppressive therapy.

59 BASIC BIOLOGICAL SCIENCES↗