scholarly journals Murine gammaherpesvirus 68 bcl-2 homologue contributes to latency establishment in vivo

2005 ◽  
Vol 86 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Brigitte D. de Lima ◽  
Janet S. May ◽  
Sofia Marques ◽  
J. Pedro Simas ◽  
Philip G. Stevenson
Keyword(s):  
Cell Activation ◽  
Ex Vivo ◽  
Lytic Replication ◽  
B Cell Activation ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

The gammaherpesviruses are characteristically latent in lymphocytes and exploit lymphocyte proliferation to establish a large, persistent pool of latent genomes. Murine gammaherpesvirus 68 (MHV-68) allows the in vivo analysis of viral genes that contribute to this and other aspects of host colonization. In this study, the MHV-68 bcl-2 homologue, M11, was disrupted either in its BH1 homology domain or upstream of its membrane-localizing C-terminal domain. Each M11 mutant showed normal lytic replication in vitro and in vivo, but had a reduction in peak splenic latency. Lower infectious-centre titres correlated with lower in vivo B-cell activation, lower viral genome loads and reduced viral tRNA expression. This was therefore a true latency deficit, rather than a deficit in ex vivo reactivation. Stable, long-term levels of splenic latency were normal. M11 function therefore contributed specifically to viral latency amplification in infected lymphoid tissue.

scholarly journals The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo

2004 ◽  
Vol 78 (2) ◽  
pp. 758-767 ◽  
Author(s):  
A. C. Townsley ◽  
B. M. Dutia ◽  
A. A. Nash
Keyword(s):  
Small Animal ◽  
Lytic Replication ◽  
Productive Infection ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) infection of mice represents a viable small-animal model for the study of gammaherpesvirus pathogenesis. MHV-76 is a deletion mutant of MHV-68, which lacks four MHV-68-specific genes (M1 to M4) and eight viral tRNA-like sequences at the 5′ end of the genome. These genes are implicated in latency and/or immune evasion. Consequently, MHV-76 is attenuated in the acute phase of in vivo infection with respect to MHV-68. Little is known about the role of M4 in viral infection, except that it is expressed as an immediate-early/early transcript during lytic replication of MHV-68 in vitro. To elucidate the contribution M4 makes to in vivo pathogenesis, we created a novel MHV-76 mutant (MHV-76inM4), in which the region of MHV-68 coding for M4 and accompanying putative promoter elements were inserted into the 5′ region of the MHV-76 genome. The growth of MHV-76inM4 in vitro was indistinguishable from that of MHV-76 and MHV-68. However, virus titers from MHV-76inM4-infected BALB/c mice were significantly increased with respect to MHV-76 at early times in the lung. In addition, at days 17 and 21 postinfection, there was a significant elevation in latent viral load in splenocytes of MHV-76inM4-infected mice compared to MHV-76. Like MHV-76-infected mice, MHV-76inM4-infected mice display no evidence of overt splenomegaly, a finding characteristic of MHV-68 infection. M4 expression in vivo was detectable during productive infection in the lung and during the establishment of latency in the spleen, but in general M4 was not detectable during long-term latency (day 100 postinfection).

scholarly journals Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency duringIn VivoInfection

2015 ◽  
Vol 89 (11) ◽  
pp. 5788-5800 ◽  
Author(s):  
Jing Qi ◽  
Chuanhui Han ◽  
Danyang Gong ◽  
Ping Liu ◽  
Sheng Zhou ◽  
...  
Keyword(s):  
Lytic Replication ◽  
Laboratory Mice ◽  
Transcription Activator ◽  
Murine Gammaherpesvirus ◽  
Responsive Gene ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Lytic Genes

ABSTRACTReplication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit ofORF48from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates theORF48promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directlyin vitroand also associates with ORF48 promoterin vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE duringde novoinfection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48in vitroandin vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identifiedORF48as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication bothin vitroandin vivo.IMPORTANCEThe replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identifiedORF48as an RTA-responsive gene of MHV-68 and mapped theciselement involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infectionin vivo. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.

scholarly journals Signaling through Toll-Like Receptors Induces Murine Gammaherpesvirus 68 Reactivation In Vivo

2008 ◽  
Vol 83 (3) ◽  
pp. 1474-1482 ◽  
Author(s):  
Lisa M. Gargano ◽  
J. Craig Forrest ◽  
Samuel H. Speck
Keyword(s):  
B Cell ◽  
Cell Activation ◽  
B Cell Activation ◽  
Toll Like Receptors ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Stimulation Of

ABSTRACT Murine gammaherpesvirus 68 (MHV68) establishes a lifelong infection in mice and is used as a model pathogen to study the role of viral and host factors in chronic infection. The maintenance of chronic MHV68 infection, at least in some latency reservoirs, appears to be dependent on the capacity of the virus to reactivate from latency in vivo. However, the signals that lead to MHV68 reactivation in vivo are not well characterized. Toll-like receptors (TLRs), by recognizing the specific patterns of microbial components, play an essential role in the activation of innate immunity. In the present study, we investigated the capacity of TLR ligands to induce MHV68 reactivation, both in vitro and in vivo. The stimulation of latently infected B cell lines with ligands for TLRs 3, 4, 5, and 9 enhanced MHV68 reactivation; the ex vivo stimulation of latently infected primary splenocytes, recovered from infected mice, with poly(I:C), lipopolysaccharide, flagellin, or CpG DNA led to early B-cell activation, B-cell proliferation, and a significant increase in the frequency of latently infected cells reactivating the virus. In vivo TLR stimulation also induced B-cell activation and MHV68 reactivation, resulting in heightened levels of virus replication in the lungs which correlated with an increase in MHV68-specific CD8+ T-cell responses. Importantly, TLR stimulation also led to an increase in MHV68 latency, as evidenced by an increase in viral genome-positive cells 2 weeks post-in vivo stimulation by specific TLR ligands. Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs.

scholarly journals Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Arundhati Gupta ◽  
Darby G. Oldenburg ◽  
Eduardo Salinas ◽  
Douglas W. White ◽  
J. Craig Forrest
Keyword(s):  
Chronic Infection ◽  
Ex Vivo ◽  
Kaposi Sarcoma ◽  
Lytic Replication ◽  
Nuclear Antigen ◽  
Content Type ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo. Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo. kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo. These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68. IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

scholarly journals Murine gammaherpesvirus-68 ORF28 encodes a non-essential virion glycoprotein

2005 ◽  
Vol 86 (4) ◽  
pp. 919-928 ◽  
Author(s):  
Janet S. May ◽  
Heather M. Coleman ◽  
Jessica M. Boname ◽  
Philip G. Stevenson
Keyword(s):  
Function Analysis ◽  
Lytic Replication ◽  
Specific Gene ◽  
Virion Protein ◽  
Major Function ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

Murine gammaherpesvirus-68 (MHV-68) ORF28 is a gammaherpesvirus-specific gene of unknown function. Analysis of epitope-tagged ORF28 protein indicated that it was membrane-associated and incorporated into virions in N-glycosylated, O-glycosylated and unglycosylated forms. The extensive glycosylation of the small ORF28 extracellular domain – most forms of the protein appeared to be mainly carbohydrate by weight – suggested that a major function of ORF28 is to attach a variety of glycans to the virion surface. MHV-68 lacking ORF28 showed normal lytic replication in vitro and in vivo and normal latency establishment. MHV-68 ORF28 therefore encodes a small, membrane-bound and extensively glycosylated virion protein, whose function is entirely dispensable for normal, single-cycle host colonization.

scholarly journals ORF23 of murine gammaherpesvirus 68 is non-essential for in vitro and in vivo infection

2012 ◽  
Vol 93 (5) ◽  
pp. 1076-1080 ◽  
Author(s):  
S. Ohno ◽  
B. Steer ◽  
C. Sattler ◽  
H. Adler
Keyword(s):  
Deletion Mutant ◽  
Protein Product ◽  
Lytic Replication ◽  
Murine Gammaherpesvirus ◽  
The Face ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

Although ORF23 is conserved among gammaherpesviruses, its role during infection is unknown. Here, we studied the expression of ORF23 of murine gammaherpesvirus 68 (MHV-68) and its role during infection. ORF23 mRNA was detected in infected cells as a late transcript. The ORF23 protein product could be expressed and detected as an N-terminally FLAG-tagged protein by Western blot and indirect immunofluorescence. To investigate the role of ORF23 in the infection cycle of a gammaherpesvirus, we constructed an ORF23 deletion mutant of MHV-68. The analysis of the ORF23 deletion mutant suggested that ORF23 of MHV-68 is neither essential for replication in cell culture nor for lytic or latent infection in vivo. A phenotype of the ORF23 deletion mutant, reflected by a moderate reduction in lytic replication and latency amplification, was only detectable in the face of direct competition to the parental virus.

scholarly journals The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection

2009 ◽  
Vol 83 (16) ◽  
pp. 8163-8172 ◽  
Author(s):  
B. Flach ◽  
B. Steer ◽  
N. N. Thakur ◽  
J. Haas ◽  
H. Adler
Keyword(s):  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type ◽  
Coding Region ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Revertant Virus

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. According to the colinear organization of the γHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.

scholarly journals Murine Gammaherpesvirus 68 Open Reading Frame 11 Encodes a Nonessential Virion Component

2005 ◽  
Vol 79 (5) ◽  
pp. 3163-3168 ◽  
Author(s):  
Jessica M. Boname ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Lytic Replication ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.

scholarly journals Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency

2003 ◽  
Vol 77 (4) ◽  
pp. 2410-2417 ◽  
Author(s):  
Heather M. Coleman ◽  
Brigitte de Lima ◽  
Victoria Morton ◽  
Philip G. Stevenson
Keyword(s):  
Thymidine Kinase ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Wild Type ◽  
Intranasal Inoculation ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.

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