Functional Characterization of P3N-PIPO Protein in the Potyviral Life Cycle

Potyviruses represent the largest genus of plant-infecting viruses and include many agriculturally important viruses such as Turnip mosaic virus (TuMV), Soybean mosaic virus (SMV) and Plum pox virus (PPV). The potyviral genome consists of a large open reading frame (ORF) and a small ORF owing to a translational or transcriptional slippage in the P3 cistron. The polyproteins encoded by these two ORFs are proteolytically processed into 11 mature proteins. Recent studies have shown that P3N-PIPO, the frameshift resulting protein, is a plasmodesmata (PD)-located protein and involved in potyviral cell-to-cell movement by mediating the targeting of the potyviral CI protein to PD to form canonical structures for potyviral cell-to-cell movement. In this study, I introduced in a full-length cDNA infectious clone of TuMV a stop codon or point mutations into P3N-PIPO without affecting the amino acid sequence of other viral proteins including P3 and evaluated effects of these mutations on TuMV infection. It was found that elimination of PIPO or substitution of the positive charged amino acid lysine with the negatively charged amino acid glutamic acid within PIPO compromises TuMV cell-to-cell spreading in Nicotiana benthamiana plants. PEG-mediated transfection assay revealed that virus replication of these mutants is not affected in Nicotiana benthamiana protoplasts. Moreover, transient co-expression of CI and P3N-PIPO mutants showed that the PIPO mutants lose ability to target the TuMV CI protein to plasmodesmata. Subcellular localization of these PIPO mutants indicated that the substitution mutants retain their PD-targeting. These data strongly support that the potyviral P3N-PIPO protein is likely a dedicated protein for potyviral intercellular movement via PD

Rover: Poor (but Elegant) Man’s Testbed

International audienceThis paper presents the OpenVisualizer Rover testbed, a simple, easy-to-deploy and cheap testbed for the Internet of Things (IoT). The OpenWSN project provides a free and open-source implementation of a standards-compliant protocol stack for the IoT, as well as all the necessary network management and debugging tools. The network management software, OpenVisualizer, is portable across popular operating systems, and connects the OpenWSN low-power wireless mesh network to the Internet. In the current setup, motes are connected to the USB ports of the computer the OpenVisualizer runs on. The OpenVisualizer monitors the internal state of those motes, which it presents through a web interface. Rover extends the OpenVisualizer by allowing motes plugged into different computers to remotely connect to it. Once connected, a user monitors and manages the motes exactly as if they were connected locally. This offers endless experimentation possibilities, as the resulting testbed can be quickly (re)deployed in realistic environments. An example Rover testbed has been deployed at the Cisco Paris Innovation and Research Lab. This paper discusses the Rover architecture, the deployment, and the experimental research done with it

Self-managing cloud-native applications : design, implementation and experience

Running applications in the cloud efficiently requires much more than deploying software in virtual machines. Cloud applications have to be continuously managed: (1) to adjust their resources to the incoming load and (2) to face transient failures replicating and restarting components to provide resiliency on unreliable infrastructure. Continuous management monitors application and infrastructural metrics to provide automated and responsive reactions to failures (health management) and changing environmental conditions (auto-scaling) minimizing human intervention. In the current practice, management functionalities are provided as infrastructural or third party services. In both cases they are external to the application deployment. We claim that this approach has intrinsic limits, namely that separating management functionalities from the application prevents them from naturally scaling with the application and requires additional management code and human intervention. Moreover, using infrastructure provider services for management functionalities results in vendor lock-in effectively preventing cloud applications to adapt and run on the most effective cloud for the job. In this paper we discuss the main characteristics of cloud native applications, propose a novel architecture that enables scalable and resilient self-managing applications in the cloud, and relate on our experience in porting a legacy application to the cloud applying cloud-native principles

Medium access control, error control and routing in underwater acoustic networks: a discussion on protocol design and implementation

The journey of underwater communication which began from Leonardo’s era took four and a half centuries to find practical applications for military purposes during World War II. However, over the last three decades, underwater acoustic communications witnessed a massive development due to the advancements in the design of underwater communicating peripherals and their supporting protocols. Successively, doors are opened for a wide range of applications to employ in the underwater environment, such as oceanography, pollution monitoring, offshore exploration, disaster prevention, navigation assistance, monitoring, coastal patrol and surveillance. Different applications may have different characteristics and hence, may require different network architectures. For instance, routing protocols designed for unpartitioned multi-hop networks are not suitable for Delay-Tolerant Networks. Furthermore, single-hop networks do not need routing protocols at all. Therefore, before developing a protocol one must study the network architecture properly and design it accordingly. There are several other factors which should also be considered with the network architecture while designing an efficient protocol for underwater networks, such as long propagation delay, limited bandwidth, limited battery power, high bit error rate of the channel and several other adverse properties of the channel, such as, multi-path, fading and refractive behaviors. Moreover, the environment also has an impact on the performance of the protocols designed for underwater networks. Even temperature changes in a single day have an impact on the performance of the protocols. A good protocol designed for any network should consider some or all of these characteristics to achieve better performance. In this thesis, we first discuss the impact of the environment on the performance of MAC and routing protocols. From our investigation, we discover that even temperature changes within a day may affect the sound speed profile and hence, the channel changes and the protocol performance vary. After that we discuss several protocols which are specifically designed for underwater acoustic networks to serve different purposes and for different network architectures. Underwater Selective Repeat (USR) is an error control protocol designed to assure reliable data transmission in the MAC layer. One may suspect that employing an error control technique over a channel which already suffers from long propagation delays is a burden. However, USR utilizes long propagation by transmitting multiple packets in a single RTT using an interlacing technique. After USR, a routing protocol for surveillance networks is discussed where some sensors are laid down at the bottom of the sea and some sinks are placed outside the area. If a sensor detects an asset within its detection range, it announces the presence of intruders by transmitting packets to the sinks. It may happen that the discovered asset is an enemy ship or an enemy submarine which creates noise to jam the network. Therefore, in surveillance networks, it is necessary that the protocols have jamming resistance capabilities. Moreover, since the network supports multiple sinks with similar anycast address, we propose a Jamming Resistance multi-path Multi-Sink Routing Protocol (MSRP) using a source routing technique. However, the problem of source routing is that it suffers from large overhead (every packet includes the whole path information) with respect to other routing techniques, and also suffers from the unidirectional link problem. Therefore, another routing protocol based on a distance vector technique, called Multi-path Routing with Limited Cross-Path Interference (L-CROP) protocol is proposed, which employs a neighbor-aware multi-path discovery algorithm to support low interference multiple paths between each source-destination pair. Following that, another routing protocol is discussed for next generation coastal patrol and surveillance network, called Underwater Delay-Tolerant Network (UDTN) routing where some AUVs carry out the patrolling work of a given area and report to a shore based control-center. Since the area to be patrolled is large, AUVs experience intermittent connectivity. In our proposed protocol, two nodes that understand to be in contact with each other calculate and divide their contact duration equally so that every node gets a fair share of the contact duration to exchange data. Moreover, a probabilistic spray technique is employed to restrict the number of packet transmissions and for error correction a modified version of USR is employed. In the appendix, we discuss a framework which was designed by our research group to realize underwater communication through simulation which is used in most of the simulations in this thesis, called DESERT Underwater (short for DEsign, Simulate, Emulate and Realize Test-beds for Underwater network protocols). It is an underwater extension of the NS-Miracle simulator to support the design and implementation of underwater network protocols. Its creation assists the researchers in to utilizing the same codes designed for the simulator to employ in actual hardware devices and test in the real underwater scenario

Efficient Range and Join Query Processing in Massively Distributed Peer-to-Peer Networks

Peer-to-peer (P2P) has become a modern distributed computing architecture that supports massively large-scale data management and query processing. Complex query operators such as range operator and join operator are needed by various distributed applications, including content distribution, locality-aware services, computing resource sharing, and many others. This dissertation tackles a number of problems related to range and join query processing in P2P systems: fault-tolerant range query processing under structured P2P architecture, distributed range caching under unstructured P2P architecture, and integration of heterogeneous data under unstructured P2P architecture. To support fault-tolerant range query processing so as to provide strong performance guarantees in the presence of network churn, effective replication schemes are developed at either the overlay network level or the query processing level. To facilitate range query processing, a prefetch-based caching approach is proposed to eliminate the performance bottlenecks incurred by those data items that are not well cached in the network. Finally, a purely decentralized partition-based join query operator is devised to realize bandwidth-efficient join query processing under unstructured P2P architecture. Theoretical analysis and experimental simulations demonstrate the effectiveness of the proposed approaches

Investigation of Novel Functions for DNA Damage Response and Repair Proteins in Escherichia coli and Humans

Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER requires more than 25 proteins to efficiently recognize and incise a damaged site. XPC-RAD23B (XPC) is the damage recognition factor in eukaryotic global genome NER. The association rate of XPC to damaged DNA has been extensively studied; however, our data suggests that the dissociation of the XPC-DNA complex is the rate-limiting step in NER. The factor that verifies DNA-damage downstream of XPC is XPA. XPA also has been implicated in binding of ds-ssDNA junctions and has been found to bind at or near double-strand break sites in the premature aging syndrome Hutchinson-Gilford progeria (HGPS). This role for XPA is outside of its known function in NER and suggests that XPA may bind at collapsed replication forks in HGPS that are unprotected due to a lack of binding by replication proteins. Along with XPC and XPA, ataxia telangiectasia and Rad3-related (ATR) is activated in response to DNA damage and initiates the cell cycle checkpoint pathway to rescue cells from genomic instability. We found that ATR functions outside of its known role in the checkpoint signaling cascade. Our data demonstrate that ATR can rescue cells from apoptosis by inhibiting cytochrome c release at the mitochondria though direct interaction with the outer mitochondrial membrane and the proapoptotic protein tBid. The role of ATR in apoptosis is regulated by Pin1, which can change the structure of ATR at the backbone level. All of the results presented here suggest novel roles for DNA repair proteins in the maintenance of genome stability

Mutagenesis Occuring Following Infection with Herpes Simplex Virus and the Contribution of Virus Ribonucleotide Reductase

Herpes simplex virus type 2 (HSV-2) has been associated with cervical cancer for many years and certain regions of both the HSV-1 and the HSV-2 genome can induce transformation of tissue culture cells. However, no virus transforming protein has yet been identified and no region of viral DNA appears to be stably retained in transformed cells or tumours. This implies that continued expression of a virus protein is not required and has resulted in the proposal that HSV transforms cells by a "hit and run" mechanism. HSV infection can generate chromosomal breaks together with amplification and rearrangement of cellular genes. Additionally, infection of permissive cells with UV-inactivated HSV-1 and of non-permissive cells with HSV-2 leads to an increase in the mutation frequency of the cellular hypoxanthine-guanine phosphoribosyltransferase gene. HSV-1 was also found to increase the mutagenic frequency of a plasmid-based gene located on the shuttle vector pZ189. These mutagenic effects resemble changes produced by chemical carcinogens and may represent a mechanism for transformation which would not require the retention of viral sequences. The cause of mutations occurring in HSV-infected cells is unknown but could be connected with the destabilization of doxyribonucleoside triphosphate (dNTP) pools seen in infected cells as studies of both prokaryotic and eukaryoti systems have stressed the importance of dNTP concentrations in mediating base substitutions and misincorporations. The HSV enzyme ribonucleotide reductase has a potential role in the destabilization of cellular dNTP pools since, unlike it cellular counterpart, it is not allosterically regulated. This is supported by results which show that a mutant cellular enzyme, which was deficient in allosteric control, raised the cellular mutation rate in transfected cells. In order to study the effect of HSV-2 ribonucleotide reductase on mutagenesis, expression vectors were constructed which contained the large (RRl) and small (RR2) subunits of the HSV-2 ribonucleotide reductase under the control of the inducible mouse metallothionein promoter, which responds at the transcriptional level to heavy metals such as zinc and cadmium. These constructs were used in mutagenesis assays which involved monitoring the mutation frequencies of the cellular adenine phosphoribosyltransferase gene (aprt) in Chinese hamster ovary (CHO) cells and of the plasmid-based suppressor tRNA gene (supF) in human cells. Increasing the amount of enzyme expression by zinc-induction did not increase the mutagenesis of the marker genes in either assay suggesting that virus ribonucleotide reductase expression may not be mutagenic in HSV-infected cells. Experiments using the HSV-1 mutant viruses tsl207 and tsl222, which are temperature sensitive (ts) for RRl and RR2 respectively, support this finding since these viruses were as mutagenic as wild type HSV-1 at both the permissive (P) and the non-permissive (NPT) temperatures. Although expression of the viral ribonucleotide reductase does not appear to be the cause of increased mutagenesis in infected cells, the possibility that enzyme activity is capable of producing a mutagenic effect cannot be ruled out. The mutagenesis assays described above involved only the transient expression of the viral enzyme and the lack of any mutagenic effect might be due to inefficeincies in the transfection procedure and/or insufficient expression of the viral genes. The cellular enzyme would also be present and might counteract dNTP pool imbalances brought about by viral ribonucleotide reductase expression. To address these problems, NIH3T3 and CHO cell lines were constructed using HSV-2 ribonucleotide reductase expressing plasmids that contained the G418-resistance gene (neoR). Mutations causing morphological transformation were assayed in NIH3T3 cells while in CHO cells the aprt gene was assayed. None of the G418-resistant cell lines tested were found to express RRl or RR2 and further lines will be screened for expression before repeating the experiment. The ribonucleotide reductase constructs were used to generate transgenic mice to determine the effect of enzyme expression in an animal model. The mice did not appear to be affected by the presence of a large number (50+) of viral ribonucleotide reductase sequences. Viral ribonucleotide reductase RNA was detected in these animals, however, it did not appear to be inducible and protein could not be detected. Both HSV-1 and HSV-2 increase the spontaneous mutation frequency of the plasmid based supF gene by around 6-fold which is in agreement with values obtained by others in similar studies

Diversity, abundance and ecological function of fungi and viruses in marine sponges.

The sponge holobiont can be considered as a complex ecosystem of the host, the microbiota and the interactions between them. Sponges are known to harbour diverse microorganisms, including bacteria, archaea, eukaryotes (such as fungi) and recently viruses. However, most studies have focused on the abundance and diversity of prokaryotes, while very little is known about sponge-associated fungi and viruses. Therefore, this thesis aimed to elucidate the gaps in our current knowledge of sponge symbiosis and provide insights to their potential functions. Sponge fungal diversity was investigated using traditional cultivation and ITS amplicon sequencing over two time periods. Our results indicated that there was relatively low fungal diversity and low host-specificity in the sponges studied here, which broadly reflected the fungal community in seawater. The diversity, abundance and functional analysis of viruses associated with the microbial cell fraction were investigated through metagenomic and (meta)transcriptomic analysis. Viral metagenomes were dominated by bacteriophages from the order Caudovirales (Myoviridae, Siphoviridae and Podoviridae), while eukaryotic viruses such as Phycodnaviridae and Pandoraviruses were highly expressed. Sponges appeared to contain both variable environment-derived and sponge-specific viral assemblages. Functional gene analysis revealed common features of viruses in all host environments, such as DNA replication and virion production. Interestingly, sponge viromes also contained diverse auxiliary metabolic genes, which vary with different host environments. To elucidate virus-host dynamics, a virus-host model system was established using Ruegeria phage 67, isolated from the sponge-associated bacteria Ruegeria sp. AU67. Infection growth dynamics and phage genomic analysis suggested that the phage was pseudolysogenic. Phage infection induced drastic morphological changes in the bacterial host, which is postulated to enhance the bacteria’s resistance to phagocytosis. Thus, the virus-host interaction may be mutualistic, increasing the survival of both bacteria and phage. Chemical extracts from the host sponge were found to inhibited bacterial growth and subsequently virion production, which suggests that sponges may contain specific mechanisms to maintain a stable holobiont. Lastly, fungal pathogens and viruses infecting pathogens were detected, indicating that sponges may act as reservoirs for the survival and spread of pathogens. This has global ecological implications on the health and disease of reef ecosystems