The Intrinsic Antiviral Defense to Incoming HSV-1 Genomes Includes Specific DNA Repair Proteins and Is Counteracted by the Viral Protein ICP0

Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection

Effects of Light, Food Availability and Temperature Stress on the Function of Photosystem II and Photosystem I of Coral Symbionts

Background: Reef corals are heterotrophic coelenterates that achieve high productivity through their photosynthetic dinoflagellate symbionts. Excessive seawater temperature destabilises this symbiosis and causes corals to "bleach," lowering their photosynthetic capacity. Bleaching poses a serious threat to the persistence of coral reefs on a global scale. Despite expanding research on the causes of bleaching, the mechanisms remain a subject of debate.\ud \ud Methodology/Principal Findings: This study determined how light and food availability modulate the effects of temperature stress on photosynthesis in two reef coral species. We quantified the activities of Photosystem II, Photosystem I and whole chain electron transport under combinations of normal and stressful growth temperatures, moderate and high light levels and the presence or absence of feeding of the coral hosts. Our results show that PS1 function is comparatively robust against temperature stress in both species, whereas PS2 and whole chain electron transport are susceptible to temperature stress. In the symbiotic dinoflagellates of Stylophora pistillata the contents of chlorophyll and major photosynthetic complexes were primarily affected by food availability. In Turbinaria reniformis growth temperature was the dominant influence on the contents of the photosynthetic complexes. In both species feeding the host significantly protected photosynthetic function from high temperature stress.\ud \ud Conclusions/Significance: Our findings support the photoinhibition model of coral bleaching and demonstrate that PS1 is not a major site for thermal damage during bleaching events. Feeding mitigates bleaching in two scleractinian corals, so that reef responses to temperature stresses will likely be influenced by the coinciding availabilities of prey for the host

Large-scale comparative genomic ranking of taxonomically restricted genes (TRGs) in bacterial and archaeal genomes

BACKGROUND: Lineage-specific, or taxonomically restricted genes (TRGs), especially those that are species and strain-specific, are of special interest because they are expected to play a role in defining exclusive ecological adaptations to particular niches. Despite this, they are relatively poorly studied and little understood, in large part because many are still orphans or only have homologues in very closely related isolates. This lack of homology confounds attempts to establish the likelihood that a hypothetical gene is expressed and, if so, to determine the putative function of the protein. METHODOLOGY/PRINCIPAL FINDINGS: We have developed "QIPP" ("Quality Index for Predicted Proteins"), an index that scores the "quality" of a protein based on non-homology-based criteria. QIPP can be used to assign a value between zero and one to any protein based on comparing its features to other proteins in a given genome. We have used QIPP to rank the predicted proteins in the proteomes of Bacteria and Archaea. This ranking reveals that there is a large amount of variation in QIPP scores, and identifies many high-scoring orphans as potentially "authentic" (expressed) orphans. There are significant differences in the distributions of QIPP scores between orphan and non-orphan genes for many genomes and a trend for less well-conserved genes to have lower QIPP scores. CONCLUSIONS: The implication of this work is that QIPP scores can be used to further annotate predicted proteins with information that is independent of homology. Such information can be used to prioritize candidates for further analysis. Data generated for this study can be found in the OrphanMine at http://www.genomics.ceh.ac.uk/orphan_mine

Exploitation of Herpesviral Transactivation Allows Quantitative Reporter Gene-Based Assessment of Virus Entry and Neutralization

Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology

Unusually Long Palindromes Are Abundant in Mitochondrial Control Regions of Insects and Nematodes

BACKGROUND: Palindromes are known to be involved in a variety of biological processes. In the present investigation we carried out a comprehensive analysis of palindromes in the mitochondrial control regions (CRs) of several animal groups to study their frequency, distribution and architecture to gain insights into the origin of replication of mtDNA. METHODOLOGY/PRINCIPAL FINDINGS: Many species of Arthropoda, Nematoda, Mollusca and Annelida harbor palindromes and inverted repeats (IRs) in their CRs. Lower animals like cnidarians and higher animal groups like chordates are almost devoid of palindromes and IRs. The study revealed that palindrome occurrence is positively correlated with the AT content of CRs, and that IRs are likely to give rise to longer palindromes. CONCLUSIONS/SIGNIFICANCE: The present study attempts to explain possible reasons and gives in silico evidence for absence of palindromes and IRs from CR of vertebrate mtDNA and acquisition and retention of the same in insects. Study of CRs of different animal phyla uncovered unique architecture of this locus, be it high abundance of long palindromes and IRs in CRs of Insecta and Nematoda, or short IRs of 10–20 nucleotides with a spacer region of 12–14 bases in subphylum Chelicerata, or nearly complete of absence of any long palindromes and IRs in Vertebrata, Cnidaria and Echinodermata

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch

Arabidopsis cell expansion is controlled by a photothermal switch

In Arabidopsis, the seedling hypocotyl has emerged as an exemplar model system to study light and temperature control of cell expansion. Light sensitivity of this organ is epitomized in the fluence rate response where suppression of hypocotyl elongation increases incrementally with light intensity. This finely calibrated response is controlled by the photoreceptor, phytochrome B, through the deactivation and proteolytic destruction of phytochrome-interacting factors (PIFs). Here we show that this classical light response is strictly temperature dependent: a shift in temperature induces a dramatic reversal of response from inhibition to promotion of hypocotyl elongation by light. Applying an integrated experimental and mathematical modelling approach, we show how light and temperature coaction in the circuitry drives a molecular switch in PIF activity and control of cell expansion. This work provides a paradigm to understand the importance of signal convergence in evoking different or non-intuitive alterations in molecular signalling

Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.DFG grant He 2526/6-2.European Commission grants QLRT-2001-01112 and MRTN-CT-2005-019248.Helmholtz Association through VISTRIE VH-VI-242.UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

The human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells

Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV