Interspecific killing of wolverines by one wolf pack.

Interactions between different species of predators are not uncommon, yet they are generally understudied in North America. Across their range, gray wolves (Canis lupus) and wolverines (Gulo gulo) occupy similar habitats and dietary niches. However, due to the elusiveness and relatively low density of these two species, interactions between them are not well documented. Here, we describe three instances of a single wolf pack killing a wolverine in the span of 13 months. None of the wolverines killed by wolves were consumed, suggesting that food was not the primary motivation behind the killings. Alternatively, defense of a food resource, territoriality, interspecific competitive killing, or some combination of those behaviors appear to be the cause of these actions. Documentation of these occurrences improves our understanding of wolf and wolverine ecology, interspecific predator interactions, and potential future changes to this aspect of community ecology.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian Uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect, unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9 , and the viral DNA polymerase processivity factor, vPPF encoded by ORF59 . MHV68 vUNG colocalized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone, or in combination. Last, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE: Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo . In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus associated cancers.

Combinatorial Loss of the Enzymatic Activities of Viral Uracil-DNA Glycosylase and Viral dUTPase Impairs Murine Gammaherpesvirus Pathogenesis and Leads to Increased Recombination-Based Deletion in the Viral Genome.

Misincorporation of uracil or spontaneous cytidine deamination is a common mutagenic insult to DNA. Herpesviruses encode a viral uracil-DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and nonenzymatic functions. However, the coordinated roles of these enzymatic activities in gammaherpesvirus pathogenesis and viral genomic stability have not been defined. In addition, potential compensation by the host UNG has not been examined in vivo The genetic tractability of the murine gammaherpesvirus 68 (MHV68) system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG-/- mice compared to wild-type (WT) mice, indicating host UNG does not compensate for the absence of vUNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in replication in the lungs. However, disabling both vUNG and vDUT led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the nonessential yellow fluorescent protein (YFP) reporter gene from the viral genome, due to recombination at repetitive elements. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs.IMPORTANCE Unrepaired uracils in DNA can lead to mutations and compromise genomic stability. Herpesviruses have hijacked host processes of DNA repair and nucleotide metabolism by encoding a viral UNG that excises uracils and a viral dUTPase that initiates conversion of dUTP to dTTP. To better understand the impact of these processes on gammaherpesvirus pathogenesis, we examined the separate and collaborative roles of vUNG and vDUT upon MHV68 infection of mice. Simultaneous disruption of the enzymatic activities of both vUNG and vDUT led to a severe defect in acute replication and establishment of latency, while also revealing a novel, combinatorial function in promoting viral genomic stability. We propose that herpesviruses require these enzymatic processes to protect the viral genome from damage, possibly triggered by misincorporated uracil. This reveals a novel point of therapeutic intervention to potentially block viral replication and reduce the fitness of multiple herpesviruses.

Maintenance of influenza A viruses and antibody response in mallards (Anas platyrhynchos) sampled during the non-breeding season in Alaska.

Prevalence of influenza A virus (IAV) infections in northern-breeding waterfowl has previously been reported to reach an annual peak during late summer or autumn; however, little is known about IAV infection dynamics in waterfowl populations persisting at high-latitude regions such as Alaska, during winter. We captured mallards (Anas platyrhynchos) throughout the non-breeding season (August-April) of 2012-2015 in Fairbanks and Anchorage, the two largest cities in Alaska, to assess patterns of IAV infection and antibody production using molecular methods and a standard serologic assay. In addition, we used virus isolation, genetic sequencing, and a virus microneutralization assay to characterize viral subtypes and to evaluate the immune response of mallards captured on multiple occasions through time. We captured 923 mallards during three successive sampling years: Fairbanks in 2012/13 and 2013/14, and Anchorage in 2014/15. Prevalence varied by age, season, and year/site with high and relatively stable estimates throughout the non-breeding season. Infected birds were detected in all locations/seasons except early-winter in Fairbanks during 2013/14. IAVs with 17 combinations of hemagglutinin (H1-5, H7-9, H11, H12) and neuraminidase (N1-6, N8, N9) subtypes were isolated. Antibodies to IAVs were detected throughout autumn and winter for all sampling locations and years, however, seroprevalence was higher among adults and varied among years. Mallards exhibited individual heterogeneity with regard to immune response, providing instances of both seroconversion and seroreversion to detected viral subtypes. The probability that an individual transitioned from one serostatus to another varied by age, with juvenile mallards having higher rates of seroconversion and seroreversion than adults. Our study provides evidence that a diversity of IAVs circulate in populations of mallards wintering at urban locations in Alaska, and we suggest waterfowl wintering at high-latitudes may play an important role in maintenance of viruses across breeding seasons.

Synthesis and biological evaluation of RON-neoglycosides as tumor cytotoxins.

Cardenolides such as digitoxin have been shown to inhibit cancer cell growth, to reduce cancer metastasis, and to induce apoptosis in tumor cells. Among the most potent digitoxin-based cytotoxins identified to date are MeON-neoglycosides generated via oxyamine neoglycosylation. Here, we report our studies of oxyamine neoglycosylation aimed at facilitating the elucidation of linkage-diversified digitoxin neoglycoside structure-activity relationships. We identified conditions suitable for the convenient synthesis of digitoxin neoglycosides and found that sugar structure, rather than RON-glycosidic linkage, exerts the strongest influence on neoglycoside yield and stereochemistry. We synthesized a library of digitoxin neoglycosides and assessed their cytotoxicity against eight human cancer cell lines. Consistent with previous findings, our data show that the structure of RON-neoglycosidic linkages influences both the potency and selectivity of digitoxin neoglycosides.

Modifying the glycosidic linkage in digitoxin analogs provides selective cytotoxins.

A chemoselective reaction between oxyamines and unprotected, unactivated reducing sugars was used to construct for the first time a panel of linkage-diversified neoglycosides. This panel of digitoxin analogs included potent and selective tumor cytotoxins; cytotoxicity was dependent on the structure of the glycosidic linkage. These results validate linkage diversification through neoglycosylation as a unique and simple strategy to powerfully complement existing methods for the optimization of glycoconjugates.