Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog

In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) coliocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, BOhler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66,4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51 % (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the elose relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection

Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3.

We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses

Interaction of the 89K murine cytomegalovirus immediate-early protein with core histones

The conditions that permit the interaction of immediate-early proteins of murine cytornegalovirus (MCMV) with DNA were studied. Chromatography of extracts from infected cells on MCMV DNA cellulose and calf thymus DNA cellulose showed that pp89, the regulatory major immediate-early protein, interacts with DNA and dissociates at salt concentrations between 0.3 and 0.6 M NaCl. pp76, a cleavage product of pp89, and additional minor ie1 proteins eluted already at low ionic strength. Cellular DNA-binding factors were required for association of pp89 with DNA. These factors were identified as core histones. Chromatography of IE proteins on histone-Sepharose in the absence of DNA revealed a high-binding affinity that was resistant to 2 M NaCl. These results suggest that pp89 has no direct DNA-binding activity. A role for an amino acid sequence homology in the N-terminal region of pp89 with histone H2B in the pp89-histone-DNA Interaction is discussed

In Vivo Competence of Murine Cytomegalovirus under the Control of the Human Cytomegalovirus Major Immediate-Early Enhancer in the Establishment of Latency and Reactivation

The human cytomegalovirus (HCMV) major immediate-early enhancer has been postulated to play a pivotal role in the control of latency and reactivation. However, the absence of an animal model has obstructed a direct test of this hypothesis. Here we report on the establishment of an in vivo, experimentally tractable system for quantitatively investigating physiological functions of the HCMV enhancer. Using a neonate BALB/c mouse model, we show that a chimeric murine CMV under the control of the HCMV enhancer is competent in vivo, replicating in key organs of mice with acute CMV infection and exhibiting latency/reactivation features comparable for the most part to those of the parental and revertant viruse

The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs

The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Δie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Δie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Δie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Δie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV

The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription

The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses

oHSV Genome Editing by Means of galK Recombineering

open8noThis work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G. C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.embargoed_20201017Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-FiumeLaura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fium

Numerical Study of Magnetoaerodynamic Flow Around a Hemisphere

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83560/1/AIAA-49278-455.pd