Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

Isolation of Cell Nuclei Using Inert Macromolecules to Mimic the Crowded Cytoplasm

Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 µM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media

NSs Encoded by Groundnut Bud Necrosis Virus Is a Bifunctional Enzyme

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism

Do marine phytoplankton follow Bergmann's rule sensu lato?

Global warming has revitalized interest in the relationship between body size and temperature, proposed by Bergmann's rule 150 years ago, one of the oldest manifestations of a ‘biogeography of traits’. We review biogeographic evidence, results from clonal cultures and recent micro- and mesocosm experiments with naturally mixed phytoplankton communities regarding the response of phytoplankton body size to temperature, either as a single factor or in combination with other factors such as grazing, nutrient limitation, and ocean acidification. Where possible, we also focus on the comparison between intraspecific size shifts and size shifts resulting from changes in species composition. Taken together, biogeographic evidence, community-level experiments and single-species experiments indicate that phytoplankton average cell sizes tend to become smaller in warmer waters, although temperature is not necessarily the proximate environmental factor driving size shifts. Indirect effects via nutrient supply and grazing are important and often dominate. In a substantial proportion of field studies, resource availability is seen as the only factor of relevance. Interspecific size effects are greater than intraspecific effects. Direct temperature effects tend to be exacerbated by indirect ones, if warming leads to intensified nutrient limitation or copepod grazing while ocean acidification tends to counteract the temperature effect on cell size in non-calcifying phytoplankton. We discuss the implications of the temperature-related size trends in a global-warming context, based on known functional traits associated with phytoplankton size. These are a higher affinity for nutrients of smaller cells, highest maximal growth rates of moderately small phytoplankton (ca. 102 µm3), size-related sensitivities for different types of grazers, and impacts on sinking rates. For a phytoplankton community increasingly dominated by smaller algae we predict that: (i) a higher proportion of primary production will be respired within the microbial food web; (ii) a smaller share of primary production will be channeled to the classic phytoplankton – crustacean zooplankton – fish food chain, thus leading to decreased ecological efficiency from a fish-production point of view; (iii) a smaller share of primary production will be exported through sedimentation, thus leading to decreased efficiency of the biological carbon pump

Between Hype and Understatement: Reassessing Cyber Risks as a Security Strategy

Most of the actions that fall under the trilogy of cyber crime, terrorism,and war exploit pre-existing weaknesses in the underlying technology.Because these vulnerabilities that exist in the network are not themselvesillegal, they tend to be overlooked in the debate on cyber security. A UKreport on the cost of cyber crime illustrates this approach. Its authors chose to exclude from their analysis the costs in anticipation of cyber crime, such as insurance costs and the costs of purchasing anti-virus software on the basis that "these are likely to be factored into normal day-to-day expenditures for the Government, businesses, and individuals. This article contends if these costs had been quantified and integrated into the cost of cyber crime, then the analysis would have revealed that what matters is not so much cyber crime, but the fertile terrain of vulnerabilities that unleash a range of possibilities to whomever wishes to exploit them. By downplaying the vulnerabilities, the threats represented by cyber war, cyber terrorism, and cyber crime are conversely inflated. Therefore, reassessing risk as a strategy for security in cyberspace must include acknowledgment of understated vulnerabilities, as well as a better distributed knowledge about the nature and character of the overhyped threats of cyber crime, cyber terrorism, and cyber war

Accurate Strand-Specific Quantification of Viral RNA

The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region