Edward Z. Yang

Human cytomegalovirus expresses novel microRNAs during productive viral infection

MicroRNAs (miRNAs) are a large class of ∼22‐nucleotide non‐coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non‐immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the hosts gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory‐adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus‐encoded miRNAs may be more common than previously believed.

A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

Eliminating Cache-Based Timing Attacks with Instruction-Based Scheduling

Information flow control allows untrusted code to access sensitive and trustworthy information without leaking this information. However, the presence of covert channels subverts this security mechanism, allowing processes to communicate information in violation of IFC policies. In this paper, we show that concurrent deterministic IFC systems that use time-based scheduling are vulnerable to a cache-based internal timing channel. We demonstrate this vulnerability with a concrete attack on Hails, one particular IFC web framework. To eliminate this internal timing channel, we implement instruction-based scheduling, a new kind of scheduler that is indifferent to timing perturbations from underlying hardware components, such as the cache, TLB, and CPU buses. We show this scheduler is secure against cache-based internal timing attacks for applications using a single CPU. To show the feasibility of instruction-based scheduling, we have implemented a version of Hails that uses the CPU retired-instruction counters available on commodity Intel and AMD hardware. We show that instruction-based scheduling does not impose significant performance penalties. Additionally, we formally prove that our modifications to Hails’ underlying IFC system preserve non-interference in the presence of caches.

mXSS attacks: attacking well-secured web-applications by using innerHTML mutations

Back in 2007, Hasegawa discovered a novel Cross-Site Scripting (XSS) vector based on the mistreatment of the backtick character in a single browser implementation. This initially looked like an implementation error that could easily be fixed. Instead, as this paper shows, it was the first example of a new class of XSS vectors, the class of mutation-based XSS (mXSS) vectors, which may occur in innerHTML and related properties. mXSS affects all three major browser families: IE, Firefox, and Chrome. We were able to place stored mXSS vectors in high-profile applications like Yahoo! Mail, Rediff Mail, OpenExchange, Zimbra, Roundcube, and several commercial products. mXSS vectors bypassed widely deployed server-side XSS protection techniques (like HTML Purifier, kses, htmlLawed, Blueprint and Google Caja), client-side filters (XSS Auditor, IE XSS Filter), Web Application Firewall (WAF) systems, as well as Intrusion Detection and Intrusion Prevention Systems (IDS/IPS). We describe a scenario in which seemingly immune entities are being rendered prone to an attack based on the behavior of an involved party, in our case the browser. Moreover, it proves very difficult to mitigate these attacks: In browser implementations, mXSS is closely related to performance enhancements applied to the HTML code before rendering; in server side filters, strict filter rules would break many web applications since the mXSS vectors presented in this paper are harmless when sent to the browser. This paper introduces and discusses a set of seven different subclasses of mXSS attacks, among which only one was previously known. The work evaluates the attack surface, showcases examples of vulnerable high-profile applications, and provides a set of practicable and low-overhead solutions to defend against these kinds of attacks.

A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL.

Significance Mapping neutralizing epitopes on viral entry glycoproteins allows the identification of potentially important functional regions. The structure of varicella-zoster virus (VZV) gHgL bound to two antibodies isolated from immune donors reveals a common binding site. Functional experiments demonstrate that the two antibodies neutralize VZV infection and inhibit glycoprotein gB/glycoprotein complex gHgL-mediated membrane fusion. Immunization experiments in mice demonstrate that VZV gHgL elicits potently neutralizing antibodies and confirm the key role of this antigenic site in antibody-mediated virus neutralization. This manuscript sheds light on the molecular mechanism of herpesvirus cell entry and will guide the design of subunit-based vaccines against VZV. Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

The cytoplasmic domain of varicella-zoster virus glycoprotein H regulates syncytia formation and skin pathogenesis.

The conserved herpesvirus fusion complex consists of glycoproteins gB, gH, and gL which is critical for virion envelope fusion with the cell membrane during entry. For Varicella Zoster Virus (VZV), the complex is necessary for cell-cell fusion and presumed to mediate entry. VZV causes syncytia formation via cell-cell fusion in skin and in sensory ganglia during VZV reactivation, leading to neuronal damage, a potential contributory factor for the debilitating condition of postherpetic neuralgia. The gH cytoplasmic domain (gHcyt) is linked to the regulation of gB/gH-gL-mediated cell fusion as demonstrated by increased cell fusion in vitro by an eight amino acid (aa834-841) truncation of the gHcyt. The gHcyt regulation was identified to be dependent on the physical presence of the domain, and not of specific motifs or biochemical properties as substitution of aa834-841 with V5, cMyc, and hydrophobic or hydrophilic sequences did not affect fusion. The importance of the gHcyt length was corroborated by stepwise deletions of aa834-841 causing incremental increases in cell fusion, independent of gH surface expression and endocytosis. Consistent with the fusion assay, truncating the gHcyt in the viral genome caused exaggerated syncytia formation and significant reduction in viral titers. Importantly, infection of human skin xenografts in SCID mice was severely impaired by the truncation while maintaining the gHcyt length with the V5 substitution preserved typical replication in vitro and in skin. A role for the gHcyt in modulating the functions of the gB cytoplasmic domain (gBcyt) is proposed as the gHcyt truncation substantially enhanced cell fusion in the presence of the gB[Y881F] mutation. The significant reduction in skin infection caused by hyperfusogenic mutations in either the gHcyt or gBcyt demonstrates that both domains are critical for regulating syncytia formation and failure to control cell fusion, rather than enhancing viral spread, is severely detrimental to VZV pathogenesis.

Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.

Mass spectrometry-based quantitative proteomic analysis of Salmonella enterica serovar Enteritidis protein expression upon exposure to hydrogen peroxide.

BackgroundSalmonellaenterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H2O2), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H2O2 or to host cells in vivo during the established phase of systemic infection have not been extensively studied.ResultsUsing stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of Salmonellaenterica serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H2O2. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H2O2, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice.ConclusionsWe have identified Salmonella proteins whose expression is modulated in the presence of H2O2. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis in vivo. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of Salmonella under oxidative stress and during its systemic infection in vivo.

IFC Inside: Retrofitting Languages with Dynamic Information Flow Control

Many important security problems in JavaScript, such as browser extension security, untrusted JavaScript libraries and safe integration of mutually distrustful websites mash-ups, may be effectively addressed using an efficient implementation of information flow control IFC. Unfortunately existing fine-grained approaches to JavaScript IFC require modifications to the language semantics and its engine, a non-goal for browser applications. In this work, we take the ideas of coarse-grained dynamic IFC and provide the theoretical foundation for a language-based approach that can be applied to any programming language for which external effects can be controlled. We then apply this formalism to server- and client-side JavaScript, show how it generalizes to the C programming language, and connect it to the Haskell LIO system. Our methodology offers design principles for the construction of information flow control systems when isolation can easily be achieved, as well as compositional proofs for optimized concrete implementations of these systems, by relating them to their isolated variants.

Human Cytomegalovirus Primase UL70 Specifically Interacts with Cellular Factor Snapin

ABSTRACT Genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV). HCMV UL70 is believed to encode the primase of the DNA replication machinery, a function which requires localization in the nucleus, the site of viral DNA synthesis. No host factors that interact with UL70 have been reported. In this study, we provide the first direct evidence that UL70 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and is associated with cellular vesicles. The interaction between UL70 and Snapin was identified in both the two-hybrid screen in yeast and coimmunoprecipitation in human cells. The nuclear import of UL70 was decreased in cells overexpressing Snapin and increased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. In contrast, no significant difference in the nuclear level of UL70, viral DNA synthesis, and progeny production was found among the parental cells and cells that either expressed a control empty vector or were treated with control siRNA molecules that did not recognize any viral or cellular transcripts. Our results suggest that Snapin may play a key role in regulating the cellular localization of UL70 in HCMV, leading to modulation of viral DNA synthesis and progeny production.