Immunogenicity evaluation of a DNA vaccine expressing the hepatitis C virus non-structural protein 2 gene in C57BL/6 Mice

Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses

Epoxide based inhibitors of the hepatitis C virus non-structural 2 autoprotease

Hepatitis C virus (HCV) non-structural 2 (NS2) encodes an essential protease activity responsible for processing at the NS2-NS3 junction which represents an attractive antiviral target. Attempts to inhibit the NS2 autoprotease with mechanism-based protease inhibitors and substrate peptides have had limited success. We report a series of epoxide-containing small molecules capable of blocking NS2-NS3 proteolysis in vitro and demonstrate the potential for selectivity towards the NS2 autoprotease. A compound within this series was able to perturb HCV genome replication in a subgenomic replicon system only when polyprotein processing was dependent on NS2 autoprotease activity, in addition it inhibited replication of full length HCV. These findings suggest blocking HCV polyprotein processing through inhibition of the NS2 autoprotease represents a viable route to exert an antiviral effect

WIRELESS NETWORK STUDY AND ANALYSIS USING NS2 SIMULATOR

NS2 (Network Simulation version 2) is a well-known generic network simulator. Unlike other expensive simulation software, it is free and based on open source. It is widely used to simulate and emulate communication networks. Furthermore, it has a rich library of network and protocol objects, which almost involve most of the aspects of network technology. This makes NS2 the most favorable simulation software which is widely used in academic research. On the other hand, the results of the simulation are validated by many research centers. For this reason many published articles about network technology show their results by using NS2 simulation. Additionally, act an excellent instruction tool NS2 is widely utilized in education. Nowadays, NS2 becomes more and more popular in scientific research and education. Nevertheless, NS2 is quite difficult to handle for a beginner. Some reasons are: the content of NS2 is very huge; the official NS manual is not updated regularly and a lot of relative knowledge and tools are involved to operate NS2 efficiently. NS2 will be one of the main tools in the research activities of the Telecommunication Engineering Group (TEG). Hence, the main target of this thesis is to study NS2 deeply and to show how to construct an emulation environment by using NS2 and MATLAB. Different simulators are given to demonstrate how to proceed with NS2. This thesis will be one reference for TEG researches for the applications of NS2.fi=Opinnäytetyö kokotekstinä PDF-muodossa.|en=Thesis fulltext in PDF format.|sv=Lärdomsprov tillgängligt som fulltext i PDF-format

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly

BACKGROUND: Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function. RESULTS: Our data revealed that expression of full-length NS2 was sufficient for the formation of inclusion bodies (IBs) that were morphologically similar to the VIBs formed during BTV infection. By using either, individual BTV proteins or infectious virions, we found that while the VP3 of the inner capsid (termed as "core") that surrounds the transcription complex was closely associated with both NS2 IBs and BTV VIBs, the surface core protein VP7 co-localized with NS2 IBs only in the presence of VP3. In contrast to the inner core proteins, the outer capsid protein VP2 was not associated with either IBs or VIBs. Like the core proteins, newly synthesized BTV RNAs also accumulated in VIBs. Unlike full-length NS2, neither the amino-, nor carboxyl-terminal fragments formed complete IB structures and each appeared to interfere in overall virus replication when similarly expressed. CONCLUSION: Together, these data demonstrate that NS2 is sufficient and necessary for IB formation and a key player in virus replication and core assembly. Perturbation of NS2 IB formation resulted in reduced virus synthesis and both the N terminal (NS2-1) and C terminal (NS2-2) fragments act as dominant negative mutants of NS2 function

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.Peer reviewe

Lineage A betacoronavirus NS2 proteins and the homologous torovirus Berne pp1a carboxy-terminal domain are phosphodiesterases that antagonize activation of RNase L

Viruses in the family Coronaviridae, within the order Nidovirales, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory diseases in humans. These viruses encode conserved replicase and structural proteins as well as more diverse accessory proteins, encoded in the 3′ ends of their genomes, that often act as host cell antagonists. We previously showed that 2′,5′-phosphodiesterases (2′,5′-PDEs) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), and by Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate-RNase L (OAS-RNase L) pathway. Here we report that additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses infecting both humans and animals, encode 2′,5′-PDEs capable of antagonizing RNase L. We used a chimeric MHV system (MHV(Mut)) in which exogenous PDEs were expressed from an MHV backbone lacking the gene for a functional NS2 protein, the endogenous RNase L antagonist. With this system, we found that 2′,5′-PDEs encoded by the human coronavirus HCoV-OC43 (OC43; an agent of the common cold), human enteric coronavirus (HECoV), equine coronavirus (ECoV), and equine torovirus Berne (BEV) are enzymatically active, rescue replication of MHV(Mut) in bone marrow-derived macrophages, and inhibit RNase L-mediated rRNA degradation in these cells. Additionally, PDEs encoded by OC43 and BEV rescue MHV(Mut) replication and restore pathogenesis in wild-type (WT) B6 mice. This finding expands the range of viruses known to encode antagonists of the potent OAS-RNase L antiviral pathway, highlighting its importance in a range of species as well as the selective pressures exerted on viruses to antagonize it. IMPORTANCE Viruses in the family Coronaviridae include important human and animal pathogens, including the recently emerged viruses severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). We showed previously that two viruses within the genus Betacoronavirus, mouse hepatitis virus (MHV) and MERS-CoV, encode 2′,5′-phosphodiesterases (2′,5′-PDEs) that antagonize the OAS-RNase L pathway, and we report here that these proteins are furthermore conserved among additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses, suggesting that they may play critical roles in pathogenesis. As there are no licensed vaccines or effective antivirals against human coronaviruses and few against those infecting animals, identifying viral proteins contributing to virulence can inform therapeutic development. Thus, this work demonstrates that a potent antagonist of host antiviral defenses is encoded by multiple and diverse viruses within the family Coronaviridae, presenting a possible broad-spectrum therapeutic target

Analysis of Multiple Flows using Different High Speed TCP protocols on a General Network

We develop analytical tools for performance analysis of multiple TCP flows (which could be using TCP CUBIC, TCP Compound, TCP New Reno) passing through a multi-hop network. We first compute average window size for a single TCP connection (using CUBIC or Compound TCP) under random losses. We then consider two techniques to compute steady state throughput for different TCP flows in a multi-hop network. In the first technique, we approximate the queues as M/G/1 queues. In the second technique, we use an optimization program whose solution approximates the steady state throughput of the different flows. Our results match well with ns2 simulations.Comment: Submitted to Performance Evaluatio