Eva Maria Borst

Identification and Expression of Human Cytomegalovirus Transcription Units Coding for Two Distinct Fcγ Receptor Homologs

ABSTRACT Cellular receptors for the Fc domain of immunoglobulin G (IgG) (FcγRs) comprise a family of surface receptors on immune cells connecting humoral and cellular immune responses. Several herpesviruses induce FcγR activities in infected cells. Here we identify two distinct human cytomegalovirus (HCMV)-encoded vFcγR glycoproteins of 34 and 68 kDa. A panel of HCMV strains exhibited a slight molecular microheterogeneity between Fcγ-binding proteins, suggesting their viral origin. To locate the responsible genes within the HCMV genome, a large set of targeted HCMV deletion mutants was constructed. The mutant analysis allowed the identification of a spliced UL119-UL118 mRNA to encode vFcγR gp68 and TRL11/IRL11 to encode vFcγR gp34. Both vFcγRs are surface resident type I transmembrane glycoproteins. Significant relatedness of sequences in the extracellular chain of gpUL119-118 and gpTRL11 with particular immunoglobulin supergene family domains present in FcγR I and FcγRs II/III, respectively, indicates a different ancestry and function of gpUL119-118 and gpTRL11. The HCMV-encoded vFcγRs highlight an impressive diversification and redundancy of FcγR structures.

Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17+)

ABSTRACT To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17+)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17+)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17+)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.

Identification of a mouse cytomegalovirus gene selectively targeting CD86 expression on antigen-presenting cells.

ABSTRACT We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti, Nat. Immunol. 2:1077-1084, 2001; S. Mathys, T. Schroeder, J. Ellwart, U. H. Koszinowski, M. Messerle, and U. Just, J. Infect. Dis. 187:988-999, 2003). Phenotypically, reduced surface expression of costimulatory molecules and major histocompatibility complex molecules was detected. In order to identify the molecular basis for the observed effects, we generated MCMV mutants with large deletions of nonessential genes. The study was facilitated by the finding that a monocyte-macrophage cell line displayed similar phenotypic alterations after MCMV infection. By analyzing the expression of cell surface molecules on infected cells, we identified a mutant virus which is no longer able to downmodulate the expression of the costimulatory molecule CD86. Additional mutants with smaller deletions allowed us to pin down the responsible gene to a certain genomic region. RNA analysis led to the identification of the spliced gene m147.5, encoding a protein with 145 amino acids. Experiments with an m147.5 mutant revealed that the protein affects CD86 expression only, suggesting that additional MCMV genes are responsible for downmodulation of the other surface molecules. Identification of viral gene products interfering with functionally important proteins of antigen-presenting cells will provide the basis to dissect the complex interaction of CMV with these important cells and to evaluate the biological importance of these viral genes in vivo.

Elimination of ie1 Significantly Attenuates Murine Cytomegalovirus Virulence but Does Not Alter Replicative Capacity in Cell Culture

ABSTRACT The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the ie1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the ie1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 × 104 PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of ie1. These results demonstrate the significance of the ie1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the ie1 gene.

The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

ABSTRACT Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

Conditional and reversible disruption of essential herpesvirus proteins

Elucidating the function of essential proteins of complex pathogenic viruses is impeded by a paucity of complementing systems. By fusing a destabilizing domain of the FK506-binding protein to essential cytomegalovirus proteins, we generated virus mutants in which amounts of fusion proteins and viral growth can be regulated by the synthetic ligand shield-1. This conditional approach will greatly facilitate the analysis of gene functions of herpesviruses and viruses of other families.

The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

ABSTRACT Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

Regulation of the Transcription and Replication Cycle of Human Cytomegalovirus Is Insensitive to Genetic Elimination of the Cognate NF-κB Binding Sites in the Enhancer

ABSTRACT The role of NF-κB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-κB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-κB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.

Proteolytic Processing of Human Cytomegalovirus Glycoprotein B Is Dispensable for Viral Growth in Culture

ABSTRACT Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-ΔFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-ΔMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACΔFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the ΔFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.

Use of Bacterial Artificial Chromosomes in Generating Targeted Mutations in Human and Mouse Cytomegaloviruses

Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC‐cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.