28 research outputs found
A pioneer of yeast genetics in Croatia: Zoran Zgagaās contribution to make national research acknowledged worldwide
This study is an attempt to evaluate the pathway and the achievements of yeast genetics in Croatia. The study represents both, an authorsā review and a historical overview and therefore is of value for yeast geneticists aswell as for historians of science
A pioneer of yeast genetics in Croatia: Zoran Zgagaās contribution to make national research acknowledged worldwide
This study is an attempt to evaluate the pathway and the achievements of yeast genetics in Croatia. The study represents both, an authorsā review and a historical overview and therefore is of value for yeast geneticists aswell as for historians of science
Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97
Abstract: The nuclear phase of herpesvirus replication is regulated through the formation of
regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear
capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely
pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric
assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the
mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in
turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide
electron microscopy-based data demonstrating the association of both nuclear capsids and NEC
proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC
constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid
interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with
earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided
by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells,
nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with
pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking
and NEC-capsid interaction
Cytomegaloviruses Exploit Recycling Rab Proteins in the Sequential Establishment of the Assembly Compartment
Cytomegaloviruses (CMV) reorganize membranous system of the cell in order to develop
a virion assembly compartment (VAC). The development starts in the early (E) phase of
infection with the reorganization of the endosomal system and the Golgi and proceeds
to the late phase until newly formed virions are assembled and released. The events in
the E phase involve reorganization of the endosomal recycling compartment (ERC) in
a series of cellular alterations that are mostly unknown. In this minireview, we discuss
the effect of murine CMV infection on Rab proteins, master regulators of membrane
trafficking pathways, which in the cascades with their GEFs and GAPs organize the flow
of membranes through the ERC. Immunofluorescence analyzes of murine CMV infected
cells suggest perturbations of Rab cascades that operate at the ERC. Analysis of cellular
transcriptome in the course of both murine and human CMV infection demonstrates
the alteration in expression of cellular genes whose products are known to build Rab
cascades. These alterations, however, cannot explain perturbations of the ERC. Cellular
proteome data available for human CMV infected cells suggests the potential role of
RabGAP downregulation at the end of the E phase. However, the very early onset of
the ERC alterations in the course of MCMV infection indicates that CMVs exploit Rab
cascades to reorganize the ERC, which represents the earliest step in the sequential
establishment of the cVAC
BPTF inhibits NK cell activity and the abundance of natural cytotoxicity receptor co-ligands
Using syngeneic BALB/c mouse breast cancer models, we show that the chromatin remodeling subunit bromodomain PHD finger transcription factor (BPTF) suppresses natural killer (NK) cell antitumor activity in the tumor microenvironment (TME). In culture, BPTF suppresses direct natural cytotoxicity receptor (NCR) mediated NK cell cytolytic activity to mouse and human cancer cell lines, demonstrating conserved functions. Blocking mouse NCR1 in vivo rescues BPTF KD tumor weights, demonstrating its importance for the control of tumor growth. We discovered that BPTF occupies heparanase (Hpse) regulatory elements, activating its expression. Increased heparanase activity results in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans (HSPGs). Using gain and loss of function approaches we show that elevated heparanase levels suppress NK cell cytolytic activity to tumor cells in culture. These results suggest that BPTF activates heparanase expression, which in turn reduces cell surface HSPGs and NCR co-ligands, inhibiting NK cell activity. Furthermore, gene expression data from human breast cancer tumors shows that elevated BPTF expression correlates with reduced antitumor immune cell signatures, supporting conserved roles for BPTF in suppressing antitumor immunity. Conditional BPTF depletion in established mouse breast tumors enhances antitumor immunity, suggesting that inhibiting BPTF could provide a novel immunotherapy
NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development
The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-Ī³. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3Ī¶. Reduced expression of CD3Ī¶ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3Ī¶ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats
Cytomegalovirus Infection: Mouse Model
This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (MCMV). Methods are included for propagating MCMV in cell cultures and for preparing a more virulent form of MCMV from salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, a method is described for preparing the murine embryonic fibroblasts used for propagating MCMV and for the plaque assay
Repair of an Attenuated Low-Passage Murine Cytomegalovirus Bacterial Artificial Chromosome Identifies a Novel Spliced Gene Essential for Salivary Gland Tropism
ABSTRACT
The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3ā² untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3ā² end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow
studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs.
Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated
, with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit
growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation