Proteomic profiling of EBNA1-host protein interactions in latent and lytic Epstein-Barr virus infections

The Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV) is expressed in both latent and lytic modes of EBV infection and contributes to EBV-associated cancers. Using a proteomics approach, we profiled EBNA1-host protein interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. We identified several interactions that occur in both modes of infection, including a previously unreported interaction with nucleophosmin and RNA-mediated interactions with several heterogeneous ribonucleoproteins (hnRNPs) and La protein.     

Proteomic profiling of the human cytomegalovirus UL35 gene products reveals a role for UL35 in the DNA repair response

Human cytomegalovirus infections involve the extensive modification of host cell pathways, including cell cycle control, the regulation of the DNA damage response, and averting promyelocytic leukemia (PML)-mediated antiviral responses. The UL35 gene from human cytomegalovirus is important for viral gene expression and efficient replication and encodes two proteins, UL35 and UL35a, whose mechanism of action is not well understood. Here, affinity purification coupled with mass spectrometry was used to identify previously unknown human cellular targets of UL35 and UL35a. We demonstrate that both viral proteins interact with the ubiquitin-specific protease USP7, and that UL35 expression can alter USP7 subcellular localization. In addition, UL35 (but not UL35a) was found to associate with three components of the Cul4(DCAF1) E3 ubiquitin ligase complex (DCAF1, DDB1, and DDA1) previously shown to be targeted by the HIV-1 Vpr protein. The coimmunoprecipitation and immunofluorescence microscopy of DCAF1 mutants revealed that the C-terminal region of DCAF1 is required for association with UL35 and mediates the dramatic relocalization of DCAF1 to UL35 nuclear bodies, which also contain conjugated ubiquitin. As previously reported for the Vpr-DCAF1 interaction, UL35 (but not UL35a) expression resulted in the accumulation of cells in the G(2) phase of the cell cycle, which is typical of a DNA damage response, and activated the G(2) checkpoint in a DCAF1-dependent manner. In addition, UL35 (but not UL35a) induced γ-H2AX and 53BP1 foci, indicating the activation of DNA damage and repair responses. Therefore, the identified interactions suggest that UL35 can contribute to viral replication through the manipulation of host responses.     

Nested Canalyzing Depth and Network Stability

We introduce the nested canalyzing depth of a function, which measures the extent to which it retains a nested canalyzing structure. We characterize the structure of functions with a given depth and compute the expected activities and sensitivities of the variables. This analysis quantifies how canalyzation leads to higher stability in Boolean networks. It generalizes the notion of nested canalyzing functions (NCFs), which are precisely the functions with maximum depth. NCFs have been proposed as gene regulatory network models, but their structure is frequently too restrictive and they are extremely sparse. We find that functions become decreasingly sensitive to input perturbations as the canalyzing depth increases, but exhibit rapidly diminishing returns in stability. Additionally, we show that as depth increases, the dynamics of networks using these functions quickly approach the critical regime, suggesting that real networks exhibit some degree of canalyzing depth, and that NCFs are not significantly better than functions of sufficient depth for many applications of the modeling and reverse engineering of biological networks.     

Up to the challenge? Hormonal and behavioral responses of free-ranging male Cassin's Sparrows,Peucaea cassinii,to conspecific song playback

The Challenge Hypothesis postulates that male vertebrates can respond to social challenges, such as simulated territorial intrusions, by rapidly increasing their concentrations of plasma androgens, such as testosterone (T). This increase may facilitate the expression of aggressive behavior and lead to persistence of this behavior even after withdrawal of the challenge, thus potentially promoting territoriality and the probability of winning future challenges. The scope of the Challenge Hypothesis was tested by exposing free-ranging male Cassin's Sparrows, Peucaea cassinii, to conspecific song playback (SPB) at the beginning of the vernal nesting season. Exposure to SPB stimulated aggressive behavior but did not influence plasma T. Furthermore, plasma T did not correlate with the duration of exposure to SPB, and the behavioral response to SPB did not differ in males that were challenged a second time shortly after the first challenge. As birds were investigated at a stage of their reproductive cycle when plasma T is presumably seasonally high due to photostimulation, the lack of hormonal response to SPB may have been due to the hypothalamus-pituitary-gonadal axis secreting hormones at maximum rates. This was not the case, however, because administration of gonadotropin-releasing hormone I rapidly stimulated the secretion of luteinizing hormone (LH) and T, and treatment with ovine LH rapidly stimulated T secretion.     

p21, an important mediator of quiescence during pituitary tumor formation, is dispensable for normal pituitary development during embryogenesis

A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components.     

Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50μM, equipotent to acyclovir.     

Vitamin K epoxide reductase contributes to protein disulfide formation and redox homeostasis within the endoplasmic reticulum

The transfer of oxidizing equivalents from the endoplasmic reticulum (ER) oxidoreductin (Ero1) oxidase to protein disulfide isomerase is an important pathway leading to disulfide formation in nascent proteins within the ER. However, Ero1-deficient mouse cells still support oxidative protein folding, which led to the discovery that peroxiredoxin IV (PRDX4) catalyzes a parallel oxidation pathway. To identify additional pathways, we used RNA interference in human hepatoma cells and evaluated the relative contributions to oxidative protein folding and ER redox homeostasis of Ero1, PRDX4, and the candidate oxidants quiescin-sulfhydryl oxidase 1 (QSOX1) and vitamin K epoxide reductase (VKOR). We show that Ero1 is primarily responsible for maintaining cell growth, protein secretion, and recovery from a reductive challenge. We further show by combined depletion with Ero1 that PRDX4 and, for the first time, VKOR contribute to ER oxidation and that depletion of all three activities results in cell death. Of importance, Ero1, PRDX4, or VKOR was individually capable of supporting cell viability, secretion, and recovery after reductive challenge in the near absence of the other two activities. In contrast, no involvement of QSOX1 in ER oxidative processes could be detected. These findings establish VKOR as a significant contributor to disulfide bond formation within the ER.