Gabriele Hahn

Publications of Gabriele Hahn

• Deletion mutant of human cytomegalovirus lacking US2-US6 and US11 maintains MHC class I expression and antigen presentation by infected dendritic cells.

  Authors: Susanne Schempp, Max Topp, Tobias Kessler, Kerstin Laib Sampaio, Kevin M Dennehy, Hermann Einsele, Gabriele Hahn, Götz Ulrich Grigoleit, Gerhard Jahn

  Virus research. 02/2011; 155(2):446-54.

  A HCMV mutant of endothelial- and DC-tropic strain TB40/E lacking the described MHC downregulating genes US2-6 and US11 (RVTB40/E(4)ΔUS11) was generated. We analyzed the susceptibility of DC toA HCMV mutant of endothelial- and DC-tropic strain TB40/E lacking the described MHC downregulating genes US2-6 and US11 (RVTB40/E(4)ΔUS11) was generated. We analyzed the susceptibility of DC to RVTB40/E(4)ΔUS11 and subsequently studied antigen presentation and T-cell stimulation. Wildtype TB40/E- and RVTB40/E(4)ΔUS11 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/E(4)ΔUS11-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/E(4)ΔUS11-infected DC efficiently stimulated CD8+ T-cells to produce IFN-γ.

• A human cytomegalovirus deleted of internal repeats replicates with near wild type efficiency but fails to undergo genome isomerization.

  Authors: Anne Sauer, Jian Ben Wang, Gabriele Hahn, Michael A McVoy

  Virology. 03/2010; 401(1):90-5.

  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 fourThe 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.

• Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.

  Authors: Christian Sinzger, Gabriele Hahn, Margarete Digel, Ruth Katona, Kerstin Laib Sampaio, Martin Messerle, Hartmut Hengel, Ulrich Koszinowski, Wolfram Brune, Barbara Adler

  The Journal of general virology. 03/2008; 89(Pt 2):359-68.

  Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not beenHuman cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

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• Dendritic-cell infection by human cytomegalovirus is restricted to strains carrying functional UL131-128 genes and mediates efficient viral antigen presentation to CD8+ T cells.

  Authors: Giuseppe Gerna, Elena Percivalle, Daniele Lilleri, Laura Lozza, Chiara Fornara, Gabriele Hahn, Fausto Baldanti, M Grazia Revello

  The Journal of general virology. 03/2005; 86(Pt 2):275-84.

  Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131-128Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131-128 genes. Here it is documented that the same genetic determinants of HCMV are responsible for monocyte-derived dendritic-cell (DC) tropism, i.e. all endotheliotropic and leukotropic strains of HCMV are also DC-tropic (or dendrotropic). In fact, all recent clinical HCMV isolates and deletion mutants sparing the UL131-128 locus as well as the endotheliotropic revertants AD169 and Towne were able to productively infect DC following co-culture with infected endothelial cells. On the contrary, the same clinical isolates extensively propagated in human fibroblasts, the UL131-128 deletion mutants and the reference laboratory strains were not. Peak extracellular virus titres in DC were reached 4-7 days post-infection (p.i.). Viral proteins pp65 and p72 were detected 1-3 h p.i., involving the great majority of DC 24 h p.i., while gB was abundantly detected 96 h p.i., when a cytopathic effect first appeared. Infection of DC with cell-free virus released into the medium could only be achieved with HCMV strains extensively adapted to growth in endothelial cells, reaching the peak titres 10 days p.i. DC infected for 24 h with cell-free virus and incubated for 16 h with autologous peripheral blood mononuclear cells were found to act as a potent stimulator of both HCMV-specific CD4+- and CD8+-mediated immune responses, as determined by cytokine flow cytometry. DC incubated with inactivated crude whole viral antigen preparations were only capable of eliciting a significant CD4+-mediated immune response.

• Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes.

  Authors: Gabriele Hahn, Maria Grazia Revello, Marco Patrone, Elena Percivalle, Giulia Campanini, Antonella Sarasini, Markus Wagner, Andrea Gallina, Gabriele Milanesi, Ulrich Koszinowski, Fausto Baldanti, Giuseppe Gerna

  Journal of virology. 10/2004; 78(18):10023-33.

  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. HCMVHuman 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.

• The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection.

  Authors: Marco Patrone, Elena Percivalle, Massimiliano Secchi, Loretta Fiorina, Guido Pedrali-Noy, Monica Zoppé, Fausto Baldanti, Gabriele Hahn, Ulrich H. Koszinowski, Gabriele Milanesi, Andrea Gallina

  The Journal of general virology. 01/2004; 84(Pt 12):3359-70.

  Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit. The R1 homologue, UL45,Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit. The R1 homologue, UL45, lacks many catalytic residues, and its impact on deoxyribonucleotide (dNTP) production remains unknown. Here, UL45 is shown to accumulate at late stages of infection and to be a virion tegument protein. To study UL45 function in its genome context, UL45 was disrupted by transposon insertion. The UL45-knockout (UL45-KO) mutant exhibited a growth defect in fibroblasts at a low m.o.i. and also a cell-to-cell spread defect. This did not result from a reduced dNTP supply because dNTP pools were unchanged in resting cells infected with the mutant virus. Irrespective of UL45 expression, all cellular RR subunits - S-phase RR subunits, and the p53-dependent p53R2 - were induced by infection. p53R2 was targeted to the infected cell nucleus, suggesting that HCMV diverts a mechanism normally activated by DNA damage response. Cells infected with the UL45-KO mutant were moderately sensitized to Fas-induced apoptosis relative to those infected with the parental virus. Together with the report on the UL45-KO endotheliotropic HCMV mutant (Hahn et al., J Virol 76, 9551-9555, 2002), these data suggest that UL45 does not share the prominent antiapototic role attributed to the mouse cytomegalovirus homologue M45 (Brune et al., Science 291, 303-305, 2001).

• Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique.

  Authors: Gabriele Hahn, Dietlind Rose, Markus Wagner, Sylvia Rhiel, Michael A McVoy

  Virology. 04/2003; 307(1):164-77.

  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;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 Towne(long) and Towne(short) (referred to as TownevarRIT3) (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.

• Tn7-mediated introduction of DNA sequences into bacmid-cloned cytomegalovirus genomes for rapid recombinant virus construction.

  Authors: Gabriele Hahn, Margit Jarosch, Jian Ben Wang, Carlos Berbes, Michael A McVoy

  Journal of virological methods. 03/2003; 107(2):185-94.

  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 thoseOur 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.

• The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics.

  Authors: Gabriele Hahn, Hanna Khan, Fausto Baldanti, Ulrich H. Koszinowski, M Grazia Revello, Giuseppe Gerna

  Journal of virology. 10/2002; 76(18):9551-5.

  An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and theAn endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RV Delta UL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

• Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Townelong as BACs and site-directed mutagenesis using a PCR-based technique

  Authors: Gabriele Hahn, Dietlind Rose, Markus Wagner, Sylvia Rhiel, Michael A McVoy

  Virology.

  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;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. Skaletskaya et al., 2001, 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.

• Tn7-mediated introduction of DNA sequences into bacmid-cloned cytomegalovirus genomes for rapid recombinant virus construction

  Authors: Gabriele Hahn, Margit Jarosch, Jian Ben Wang, Carlos Berbes, Michael A McVoy

  Journal of Virological Methods.

  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 thoseOur 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.