Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

Salinas, Eduardo; Byrum, Stephanie D.; Moreland, Linley E.; Mackintosh, Samuel G.; Tackett, Alan J.; Forrest, J. Craig

doi:10.1128/jvi.02022-15

Cited by 13 publications

(12 citation statements)

References 91 publications

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“…In our previous study, host proteins potentially interacting with SVCV G protein were screened through the TAP-MS system ( 20 ). HSC70 (Gene ID: NM_001110403.1) involved in the replication of several viruses was selected for further study ( 21 , 22 ).…”

Section: Resultsmentioning

confidence: 99%

HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

Shi

Gao

et al. 2021

Front. Immunol.

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As a fierce pathogen, spring viremia of carp virus (SVCV) can cause high mortality in the common carp, and its glycoprotein (G protein) is a component of the viral structure on the surface of virion, which is crucial in viral life cycle. This report adopted tandem affinity purification (TAP), mass spectrometry analysis (LC-MS/MS), immunoprecipitation, and confocal microscopy assays to identify Heat shock cognate protein 70 (HSC70) as an interaction partner of SVCV G protein. It was found that HSC70 overexpression dramatically inhibited SVCV replication, whereas its loss of functions elicited opposing effects on SVCV replication. Mechanistic studies indicate that HSC70 induces lysosomal degradation of ubiquitinated-SVCV G protein. This study further demonstrates that Membrane-associated RING-CH 8 (MARCH8), an E3 ubiquitin ligase, is critical for SVCV G protein ubiquitylation and leads to its lysosomal degradation. Furthermore, the MARCH8 mediated ubiquitylation of SVCV G protein required the participation of HSC70 through forming a multicomponent complex. Taken together, these results demonstrate that HSC70 serves as a scaffold for MARCH8 and SVCV G, which leads to the ubiquitylation and degradation of SVCV G protein and thus inhibits viral replication. These findings have established a novel host defense mechanism against SVCV.

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Section: Resultsmentioning

confidence: 99%

HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

Shi

Gao

et al. 2021

Front. Immunol.

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“…This kLANA function may be partially responsible for the "latent" phenotype of KSHV infection observed in most cell types, as repression of ORF50 by kLANA limits lytic viral gene expression in cells infected de novo (31,32). In contrast, MHV68 undergoes efficient lytic replication in most cell types, and mLANA, which is also a transcription regulator, facilitates viral replication (14,20,23,33). Given that KLKI MHV68 exhibited attenuated lytic replication and reactivation, we speculated that kLANA was able to repress the ORF50 promoter in MHV68.…”

Section: Resultsmentioning

confidence: 99%

“…There are also apparent functional differences between kLANA and mLANA. For MHV68, LANA facilitates efficient lytic replication by preventing infection-associated p53 responses that promote cell death and by recruiting heat shock cognate protein 70 (Hsc70) to the nuclei of infected cells, thereby promoting progression of replication (12,14,16,23). In contrast, KSHV LANA is thought to actively suppress lytic replication by repressing transcription of ORF50 (24,25), an essential gene that encodes the replication and transactivator protein RTA.…”

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confidence: 99%

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

Gupta

Oldenburg

Salinas

et al. 2017

J Virol

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Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation 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 and 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 kLANA repressed the MHV68 promoter for, 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 rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation 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. Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the 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 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 model system for defining evolutionarily conserved and divergent functions of LANA homologs in infection and disease.

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“…Previous studies using traditional mutagenesis approaches demonstrated that both the absence of mLANA and point mutations in the DNA binding domain of mLANA lead to a profound defect in latency establishment in the spleen after IN inoculation [ 18 , 19 , 21 ]. However, these mutations also result in defects in lytic viral replication [ 17 , 19 , 21 , 22 ]. Since lytic replication in the lung is important for MHV68 dissemination to the spleen after IN inoculation [ 40 ], it remained possible that defects in latency establishment observed with mLANA-null or mutant virus were indirect, the result of upstream defects in acute replication and dissemination.…”

Section: Discussionmentioning

confidence: 99%

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection

et al. 2018

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Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.

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Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

Cited by 13 publications

References 91 publications

HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

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

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection

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