scholarly journals Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication

2007 ◽  
Vol 81 (13) ◽  
pp. 7300-7305 ◽  
Author(s):  
Heiko Adler ◽  
Beatrix Steer ◽  
Klaus Freimüller ◽  
Jürgen Haas
Keyword(s):  
Artificial Chromosome ◽  
Plasmid Replication ◽  
Origin Of Replication ◽  
Replication Assay ◽  
Murine Gammaherpesvirus ◽  
Origins Of Replication ◽  
Dna Fragment ◽  
Gammaherpesvirus 68 ◽  
The Right ◽  
Murine Gammaherpesvirus 68

ABSTRACT A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.

scholarly journals Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats

2005 ◽  
Vol 86 (3) ◽  
pp. 561-574 ◽  
Author(s):  
Heather M. Coleman ◽  
Stacey Efstathiou ◽  
Philip G. Stevenson
Keyword(s):  
Transcription Initiation ◽  
Repeat Unit ◽  
Initiation Site ◽  
Reading Frame ◽  
Left Hand ◽  
Murine Gammaherpesvirus ◽  
Terminal Repeats ◽  
Gammaherpesvirus 68 ◽  
The Right ◽  
Murine Gammaherpesvirus 68

Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5′ non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5′ untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.

scholarly journals Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome

2000 ◽  
Vol 74 (15) ◽  
pp. 6964-6974 ◽  
Author(s):  
Heiko Adler ◽  
Martin Messerle ◽  
Markus Wagner ◽  
Ulrich H. Koszinowski
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Mouse Strains ◽  
Viral Gene ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Host Interaction ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) inEscherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked byloxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.

scholarly journals Murine Gammaherpesvirus 68 ORF52 Encodes a Tegument Protein Required for Virion Morphogenesis in the Cytoplasm

2007 ◽  
Vol 81 (18) ◽  
pp. 10137-10150 ◽  
Author(s):  
Eric Bortz ◽  
Lili Wang ◽  
Qingmei Jia ◽  
Ting-Ting Wu ◽  
Julian P. Whitelegge ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Artificial Chromosome ◽  
Tegument Protein ◽  
Flag Epitope ◽  
Murine Gammaherpesvirus ◽  
Virion Morphogenesis ◽  
Virion Envelope ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.

scholarly journals Virus Reconstituted from Infectious Bacterial Artificial Chromosome (BAC)-Cloned Murine Gammaherpesvirus 68 Acquires Wild-Type Properties In Vivo Only after Excision of BAC Vector Sequences

2001 ◽  
Vol 75 (12) ◽  
pp. 5692-5696 ◽  
Author(s):  
Heiko Adler ◽  
Martin Messerle ◽  
Ulrich H. Koszinowski
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Recombinant Virus ◽  
Artificial Chromosome ◽  
Biological Properties ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Vector Sequences ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RγHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RγHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.

scholarly journals Alpha beta-crystallin expression and presentation following infection with murine gammaherpesvirus 68

Autoimmunity ◽  
2013 ◽  
Vol 46 (6) ◽  
pp. 399-408 ◽  
Author(s):  
Vinita S. Chauhan ◽  
Daniel A. Nelson ◽  
Ian Marriott ◽  
Kenneth L. Bost
Keyword(s):  
Murine Gammaherpesvirus ◽  
Alpha Beta ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

scholarly journals Two Kinetic Patterns of Epitope-Specific CD8 T-Cell Responses following Murine Gammaherpesvirus 68 Infection

2010 ◽  
Vol 84 (6) ◽  
pp. 2881-2892 ◽  
Author(s):  
Michael L. Freeman ◽  
Kathleen G. Lanzer ◽  
Tres Cookenham ◽  
Bjoern Peters ◽  
John Sidney ◽  
...  
Keyword(s):  
T Cell ◽  
Expression Patterns ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Immune Control ◽  
Murine Gammaherpesvirus ◽  
Cell Responses ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6487-specific cells predominate early in infection and then decline rapidly, whereas ORF61524-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2b-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that γHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.

scholarly journals The Gammaherpesvirus Chemokine Binding Protein Binds to the N Terminus of CXCL8

2003 ◽  
Vol 77 (15) ◽  
pp. 8588-8592 ◽  
Author(s):  
Louise M. C. Webb ◽  
Ian Clark-Lewis ◽  
Antonio Alcami
Keyword(s):  
Receptor Binding ◽  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Structural Requirements ◽  
Murine Gammaherpesvirus ◽  
N Terminus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus 68 gene M3 encodes a chemokine binding protein (vCKBP-3) which has no sequence similarity to chemokine receptors but inhibits chemokine receptor binding and activity. We have used a panel of CXCL8 analogs to identify the structural requirements for CXCL8 to bind to vCKBP-3 in a scintillation proximity assay. Our data suggest that vCKBP-3 acts by mimicking the binding of chemokine receptors to CXCL8.

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