Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.     

White-Matter Development is Different in Bilingual and Monolingual Children: A Longitudinal DTI Study

Although numerous people grow up speaking more than one language, the impact of bilingualism on brain developing neuroanatomy is still poorly understood. This study aimed to determine whether the changes in the mean fractional-anisotropy (MFA) of language pathways are different between bilingual and monolingual children. Simultaneous-bilinguals, sequential-bilinguals and monolingual, male and female 10–13 years old children participated in this longitudinal study over a period of two years. We used diffusion tensor tractography to obtain mean fractional-anisotropy values of four language related pathways and one control bundle: 1-left-inferior-occipitofrontal fasciculus/lIFOF, 2-left-arcuate fasciculus/lAF/lSLF, 3-bundle arising from the anterior part of corpus-callosum and projecting to orbital lobe/AC-OL, 4-fibres emerging from anterior-midbody of corpus-callosum (CC) to motor cortices/AMB-PMC, 5- right-inferior-occipitofrontal fasciculus rIFOF as the control pathway unrelated to language. These values and their rate of change were compared between 3 groups. FA-values did not change significantly over two years for lAF/lSLF and AC-OL. Sequential-bilinguals had the highest degree of change in the MFA value of lIFOF, and AMB-PMC did not present significant group differences. The comparison of MFA of lIFOF yielded a significantly higher FA-value in simultaneous bilinguals compared to monolinguals. These findings acknowledge the existing difference of the development of the semantic processing specific pathway between children with different semantic processing procedure. These also support the hypothesis that age of second language acquisition affects the maturation and myelination of some language specific white-matter pathways.     

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.     

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.     

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.     

Targeted drug delivery of magnetic microbubble for abdominal aortic aneurysm: an in silico study

Biomechanics and Modeling in Mechanobiology - Targeted drug delivery (TDD) to abdominal aortic aneurysm (AAA) using a controlled and efficient approach has recently been a significant challenge. In...