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Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids.

ABSTRACT: Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.

SUBMITTER: Grzesik P

PROVIDER: S-EPMC5661877 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids.

Grzesik Peter P MacMath Derek D Henson Brandon B Prasad Sanjana S Joshi Poorval P Desai Prashant J PJ

Virus research 20170426

Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specif ...[more]

PMID: 28456575

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Project description:UnlabelledCapsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a β-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein.ImportanceSeveral capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

Project description:BackgroundAntiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions.MethodsFor KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication.ResultsWe discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly.ConclusionsOur studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

Dataset Information

Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids.

Publications

Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids.

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