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 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 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 Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Ting-Ting Wu ◽  
Hsiang-I Liao ◽  
Leming Tong ◽  
Ronika Sitapara Leang ◽  
Greg Smith ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Cultured Cells ◽  
Artificial Chromosome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Wild Type ◽  
Bac Clone ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68

Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.

scholarly journals Glycoprotein M Is an Essential Lytic Replication Protein of the Murine Gammaherpesvirus 68

2005 ◽  
Vol 79 (6) ◽  
pp. 3459-3467 ◽  
Author(s):  
Janet S. May ◽  
Susanna Colaco ◽  
Philip G. Stevenson
Keyword(s):  
Homologous Recombination ◽  
Viral Genome ◽  
Lytic Replication ◽  
Expression Cassette ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Virion Morphogenesis ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT All herpesviruses encode a homolog of glycoprotein M (gM), which appears to function in virion morphogenesis. Despite its conservation, gM is inessential for the lytic replication of alphaherpesviruses. In order to address the importance of gM in gammaherpesviruses, we disrupted it in the murine gammaherpesvirus 68 (MHV-68). The mutant virus completely failed to propagate in normally permissive fibroblasts. The defective genome was rescued by either homologous recombination to restore the wild-type gM in situ or the insertion of an ectopic, intergenic expression cassette encoding gM into the viral genome. Thus, gM was essential for the lytic replication of MHV-68.

scholarly journals The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

2007 ◽  
Vol 81 (18) ◽  
pp. 9870-9877 ◽  
Author(s):  
Sangmi Lee ◽  
Hye-Jeong Cho ◽  
Jung-Jin Park ◽  
Yong-Sun Kim ◽  
Seungmin Hwang ◽  
...  
Keyword(s):  
Virus Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Mapping Study ◽  
Murine Gammaherpesvirus ◽  
Downstream Target ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Target Promoters

ABSTRACT Our functional mapping study of murine gammaherpesvirus 68 (MHV-68, or γHV-68) revealed that a mutant harboring a transposon at the ORF49 locus (ORF49null) evidenced a highly attenuated in vitro growth. ORF49 resides adjacent to and in an opposite direction from RTA, the primary switch of the gammaherpesvirus life cycle. A FLAG-tagged ORF49 protein was able to transcomplement ORF49null, and a revertant of ORF49null restored its attenuated growth to a level comparable to that of the wild type. The FLAG-tagged ORF49 protein promoted the ability of RTA to activate downstream target promoters and enhanced virus replication from the ORF50null virus in the presence of RTA. Furthermore, ORF49 enhanced wild-type virus replication by increasing the RTA transcript levels. Our data indicate that ORF49 may perform an important function in MHV-68 replication in cooperation with RTA.

scholarly journals Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology

2004 ◽  
Vol 85 (6) ◽  
pp. 1393-1400 ◽  
Author(s):  
Bernadette M. Dutia ◽  
Douglas J. Roy ◽  
Bahram Ebrahimi ◽  
Babunilayam Gangadharan ◽  
Stacey Efstathiou ◽  
...  
Keyword(s):  
Lymph Node ◽  
Mediastinal Lymph Node ◽  
Virus Genome ◽  
Northern Analysis ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Interstitial Pulmonary Fibrosis ◽  
Lymph Node Pathology ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

Infection with the murine gammaherpesvirus MHV-68 has profound effects on splenic and mediastinal lymph node pathology in mice which lack the interferon-γ receptor (IFN-γ R−/−). In these mice MHV-68 infection causes fibrosis and loss of lymphocytes in the spleen and the mediastinal lymph node as well as interstitial pulmonary fibrosis and fibrotic changes in the liver. The changes are associated with transient elevated latent virus loads in the spleen. Four independent virus mutants with insertions and/or deletions in the left end of the genome fail to induce the pathological changes and establish latency at normal levels in the spleen. The data indicate that the pathology does not correlate with any of the known genes encoded within this region of the genome, genes M1–M4 and the eight vtRNAs. Northern analysis of mRNAs transcribed by wild-type and mutant viruses shows that at least two uncharacterized transcripts are encoded within this region. These transcripts are absent in the mutant viruses and are candidates for the virus genes responsible for the aberrant pathology in IFN-γ R−/− mice.

scholarly journals Activation of Vav by the Gammaherpesvirus M2 Protein Contributes to the Establishment of Viral Latency in B Lymphocytes

2006 ◽  
Vol 80 (12) ◽  
pp. 6123-6135 ◽  
Author(s):  
Lénia Rodrigues ◽  
Marta Pires de Miranda ◽  
María J. Caloca ◽  
Xosé R. Bustelo ◽  
J. Pedro Simas
Keyword(s):  
B Lymphocytes ◽  
Viral Latency ◽  
Murine Gammaherpesvirus ◽  
Host Interaction ◽  
New Type ◽  
Trimolecular Complex ◽  
Gammaherpesvirus 68 ◽  
Novel Strategy ◽  
Murine Gammaherpesvirus 68

ABSTRACT Gammaherpesviruses subvert eukaryotic signaling pathways to favor latent infections in their cellular reservoirs. To this end, they express proteins that regulate or replace functionally specific signaling proteins of eukaryotic cells. Here we describe a new type of such viral-host interaction that is established through M2, a protein encoded by murine gammaherpesvirus 68. M2 associates with Vav proteins, a family of phosphorylation-dependent Rho/Rac exchange factors that play critical roles in lymphocyte signaling. M2 expression leads to Vav1 hyperphosphorylation and to the subsequent stimulation of its exchange activity towards Rac1, a process mediated by the formation of a trimolecular complex with Src kinases. This heteromolecular complex is coordinated by proline-rich and Src family-dependent phosphorylated regions of M2. Infection of Vav-deficient mice with gammaherpesvirus 68 results in increased long-term levels of latency in germinal center B lymphocytes, corroborating the importance of the M2/Vav cross talk in the process of viral latency. These results reveal a novel strategy used by the murine gammaherpesvirus family to subvert the lymphocyte signaling machinery to its own benefit.

scholarly journals Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein

2009 ◽  
Vol 83 (13) ◽  
pp. 6484-6493 ◽  
Author(s):  
Christopher M. Collins ◽  
Jeremy M. Boss ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
Virus Infection ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Viral Latency ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.

scholarly journals Kinetics of Murine Gammaherpesvirus 68 Gene Expression following Infection of Murine Cells in Culture and in Mice

2001 ◽  
Vol 75 (11) ◽  
pp. 4955-4963 ◽  
Author(s):  
Rosemary Rochford ◽  
Mary L. Lutzke ◽  
Rosiane S. Alfinito ◽  
Anaira Clavo ◽  
Rhonda D. Cardin
Keyword(s):  
Gene Expression ◽  
Rnase Protection Assay ◽  
Viral Gene ◽  
3T3 Cells ◽  
Rnase Protection ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Kinetics Of

ABSTRACT A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.

scholarly journals Induction of Protective Immunity against Murine Gammaherpesvirus 68 Infection in the Absence of Viral Latency

2009 ◽  
Vol 84 (5) ◽  
pp. 2453-2465 ◽  
Author(s):  
Qingmei Jia ◽  
Michael L. Freeman ◽  
Eric J. Yager ◽  
Ian McHardy ◽  
Leming Tong ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Lytic Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Transcription Activator ◽  
Vaccine Strategy ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.

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