Search

Summary: Death receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function. [ABSTRACT FROM AUTHOR]

Activation of the IKK/NF-κB signalling pathway is a hallmark of human cytomegalovirus (HCMV) infection. However, its role in regulating major immediate-early promoter (MIEP)-dependent transcription and HCMV replication remains controversial. This study uses a combination of genetic approaches to investigate the effects of cell culture conditions on the importance of virus-induced NF-κB activation during the infection of endothelial cells or fibroblasts. Adenoviral-mediated expression of a dominant-negative mutant of IKK2 kinase (dnIKK2) in human umbilical vein endothelial cells resulted in a strong reduction of IκBα degradation and NF-κB activation following infection with an HCMV clinical isolate. Viral replication was impaired in dnIKK2-expressing cells that were growth-arrested before infection, but not in replicating cells. The inhibitory effect of dnIKK2 was independent from the virus strain and the cell type used, because replication of the laboratory AD169 strain was impaired as well in dnIKK2-expressing quiescent fibroblasts. Moreover, progressive disruption of NF-κB response elements within the MIEP in recombinant HCMV viruses derived from the clinical isolate prevented their replication in quiescent cells but not in actively growing cells. These results demonstrate an essential role of virus-induced IKK/NF-κB activity to trigger both viral IE gene expression and productive replication in quiescent cells. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium-leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection. [ABSTRACT FROM AUTHOR]

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FiX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs. [ABSTRACT FROM AUTHOR]

The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Townelong and Towneshort (referred to asTownevarRIT3) (A. , Proc. Natl. Acad. Sci. USA 98, 7829–7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique. [Copyright &y& Elsevier]

Our basic understanding of how viruses infect, replicate, and cause disease has been largely derived from genetic approaches in which viral sequences have been mutated and the consequences of those mutations determined. In the herpesvirus field, deletions or insertions that preclude expression of specific viral proteins or remove critical cis elements have been invaluable in identifying their overall functions. We are now ready to move to a new level of detail—mapping functional domains within viral proteins or defining cis elements to the nucleotide level. This level of detail will require mutagenesis on a new scale, with recombinant viruses containing mutations within a given locus perhaps numbering in the hundreds. Mutagenesis on this scale would be greatly facilitated by more rapid methods of recombinant virus construction. In this report, we adapted a technology employing Tn7-mediated site-specific transposition [J. Virol. 67 (1993) 4566] as a rapid and highly reliable method to introduce novel sequences into bacmid-cloned herpesvirus genomes. We show that recombinant viruses can be rapidly created and that a deletion of the human cytomegalovirus (HCMV) essential gene ie2 can be complemented in cis by reintroduction, via transposition, of an ie2 cDNA; detailed mutagenesis of the complementing ie2 gene can now follow. Tn7-mediated transposition should accelerate greatly the pace at which recombinant herpesviruses can be constructed and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies of both cis- and trans-acting genetic elements. [Copyright &y& Elsevier]

Abstract: The class E genome of human cytomegalovirus (HCMV) contains long and short segments that invert due to recombination between flanking inverted repeats, causing the genome to isomerize into four distinct isomers. To determine if isomerization is important for HCMV replication, one copy of each repeat was deleted. The resulting virus replicated in cultured human fibroblasts with only a slight growth impairment. Restriction and Southern analyses confirmed that its genome is locked in the prototypic arrangement and unable to isomerize. We conclude that efficient replication of HCMV in fibroblasts does not require (i) the ability to undergo genome isomerization, (ii) genes that lie partially within the deleted repeats, or (iii) diploidy of genes that lie wholly within repeats. The simple genomic structure of this virus should facilitate studies of genome circularization, latency or persistence, and concatemer packaging as such studies are hindered by the complexities imposed by isomerization. [Copyright &y& Elsevier]

Abstract: A HCMV mutant of endothelial- and DC-tropic strain TB40/E lacking the described MHC downregulating genes US2–6 and US11 (RVTB40/E4ΔUS11) was generated. We analyzed the susceptibility of DC to RVTB40/E4ΔUS11 and subsequently studied antigen presentation and T-cell stimulation. Wildtype TB40/E- and RVTB40/E4ΔUS11 showed no significant difference in the efficiency of infection of DC. Whereas infection with TB40/E induced downregulation of MHC I, no significant MHC I downregulation was observed on RVTB40/E4ΔUS11-infected DC, indicating that the US2–6, US11 region encodes for the major genes relevant for MHC I downregulation. However, both viruses induced downregulation of MHC II, as well as CD40, CD80, CD86 and CD83 to the same levels. Stimulation of IFN-γ production by HCMV-specific CD8+ T-cells by infected autologous DC correlated with the modulation of MHC expression. While TB40/E-infected DC did not efficiently stimulate IFN-γ production, RVTB40/E4ΔUS11-infected DC efficiently stimulated CD8+ T-cells to produce IFN-γ. [ABSTRACT FROM AUTHOR]

Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus. [ABSTRACT FROM AUTHOR]

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level, vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics. [ABSTRACT FROM AUTHOR]