Murine gammaherpesvirus 68 ORF52 encodes a tegument protein required for virion morphogenesis in the cytoplasm

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.     

Distinct domains in ORF52 tegument protein mediate essential functions in murine gammaherpesvirus 68 virion tegumentation and secondary envelopment

Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologically associated with several types of human malignancies. However, as these two human gammaherpesviruses do not replicate efficiently in cultured cells, the morphogenesis of gammaherpesvirus virions is poorly understood. Murine gammaherpesvirus 68 (MHV-68) provides a tractable model to define common, conserved features of gammaherpesvirus biology. ORF52 of MHV-68 is conserved among gammaherpesviruses. We have previously shown that this tegument protein is essential for the envelopment and egress of viral particles and solved the crystal structure of ORF52 dimers. To more closely examine its role in virion maturation, we performed immunoelectron microscopy of MHV-68-infected cells and found that ORF52 localized to both mature, extracellular virions and immature viral particles in the cytoplasm. ORF52 consists of three α-helices followed by one β-strand. To understand the structural requirements for ORF52 function, we constructed mutants of ORF52 and examined their ability to complement an ORF52-null MHV-68 virus. Mutations in conserved residues in the N-terminal α1-helix and C terminus, or deletion of the α2-helix, resulted in a loss-of-function phenotype. Furthermore, the α1-helix was crucial for the predominantly punctate cytoplasmic localization of ORF52, while the α2-helix was a key domain for ORF52 dimerization. Immunoprecipitation experiments demonstrated that ORF52 interacts with another MHV-68 tegument protein, ORF42; however, a single point mutation in R95 in the C terminus of ORF52 led to the loss of this interaction. Moreover, the homologues of MHV-68 ORF52 in Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus complement the defect in ORF52-null MHV-68 and interact with MHV-68 ORF52. Taken together, these data uncover the relationship between the α-helical structure and the molecular basis for ORF52 function. This is the first structure-based functional domain mapping study for an essential gammaherpesvirus tegument protein.     

Glycoprotein M is an essential lytic replication protein of the murine gammaherpesvirus 68

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.     

The murine gammaherpesvirus 68 ORF27 gene product contributes to intercellular viral spread

Herpesviruses remain predominantly cell associated within their hosts, implying that they spread between cells by a mechanism distinct from free virion release. We previously identified the efficient release of murine gammaherpesvirus 68 (MHV-68) virions as a function of the viral gp150 protein. Here we show that the MHV-68 ORF27 gene product, gp48, contributes to the direct spread of viruses from lytically infected to uninfected cells. Monoclonal antibodies to gp48 identified it on infected cell surfaces and in virions. gp48-deficient viruses showed no obvious deficit in virion cell binding, single-cycle replication, or virion release but had reduced lytic propagation between cells. After intranasal infection of mice, ORF27-deficient viruses were impaired predominantly in lytic replication in the lungs. There was a small deficit in latency establishment, but long-term latency appeared normal. Since ORF27 has homologs in both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, it is likely part of a conserved mechanism employed by gammaherpesviruses to disseminate lytically in their hosts.     

Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.Gammaherpesviruses are associated with tumorigenesis. Like other herpesviruses, they are characterized as having two distinct stages in their life cycle: lytic replication and latency (15, 16, 18, 21, 54). Latency provides the viruses with advantages to escape host immune surveillance and to establish lifelong persistent infection and contributes to transformation and development of malignancies. However, it is through lytic replication that viruses propagate and transmit among hosts to maintain viral reservoirs. Both viral latency and lytic replication play important roles in tumorigenesis. The gammaherpesvirus subfamily includes Epstein-Barr virus (EBV), Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 and murine gammaherpesvirus 68 (MHV-68), among others. EBV is associated with Burkitt''s lymphoma, nasopharyngeal carcinoma, Hodgkin''s disease, and lymphoproliferative diseases in immunodeficient patients (28). KSHV is etiologically linked with Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease (11-13, 22, 52). Neither in vivo nor in vitro studies of EBV and KSHV are convenient due to their propensity to establish latency in cell culture and their limited host ranges.MHV-68 is genetically related to these two human gammaherpesviruses, especially to KSHV, based on the alignment of their genomic sequences and other biological properties (55). As a natural pathogen of wild rodents, MHV-68 also infects laboratory mice (6, 40, 46) and replicates to a high titer in a variety of fibroblast and epithelial cell lines. These advantages make MHV-68 an excellent model for studying the lytic replication of gammaherpesviruses in vitro and certain aspects of virus-host interactions in vivo. In addition, the MHV-68 genome has been cloned as a bacterial artificial chromosome (BAC) that can propagate in Escherichia coli (1, 2, 36, 51), making it convenient to study the function of each open reading frame (ORF) by genetic methods. Exploring the functions of MHV-68 ORFs will likely shed light on the functions of their homologues in human gammaherpesviruses.Gammaherpesviral particles have a characteristic multilayered architecture. An infectious virion contains a double-stranded DNA genome, an icosahedral capsid shell, a thick, proteinaceous tegument compartment, and a lipid bilayer envelope spiked with glycoproteins (14, 30, 47, 49). As a unique structure of herpesviruses, the tegument plays important roles in multiple aspects of the viral life cycle, including virion assembly and egress (38, 48, 53), translocation of nucleocapsids into the nucleus, transactivation of viral immediate-early genes, and modulation of host cell gene expression, innate immunity, and signal transduction (9, 10, 23, 60). Some components of MHV-68 tegument have been identified by a mass spectrometric study (8), and the functions of some tegument proteins have been revealed, such as ORF45, ORF52, and ORF75c (7, 24, 29).MHV-68 ORF33 is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. Its homologues include human herpes simplex virus type 1 (HSV-1) UL16, human herpes simplex virus type 2 (HSV-2) UL16, human cytomegalovirus (HCMV) UL94, EBV BGLF2, KSHV ORF33, and rhesus monkey rhadinovirus (RRV) ORF33. HSV-1 UL16 has been identified as a tegument protein and may function in viral DNA packaging, virion assembly, budding, and egress (5, 32, 35, 41, 44). HCMV UL94 is a virion associated protein and might function in virion assembly and budding (31, 57). EBV BGLF2, KSHV ORF33, and RRV ORF33 are also virion-associated proteins, but their functions are not clear (26, 43, 59). The mass spectrometric study of MHV-68 did not identify ORF33 as a virion component (8), although ORF33 is found to be essential for viral lytic replication by transposon mutagenesis of the MHV-68 genome cloned as a BAC (51). However, insertion of the 1.2-kbp Mu transposon in that study may influence the expression of ORFs approximate to ORF33. Consequently, the role ORF33 plays in viral replication needs to be confirmed, preferably through site-directed mutagenesis. Whether ORF33 is a tegument protein and the exact viral replication stage in which it functions also need to be investigated.We determined that MHV-68 ORF33 encodes a tegument protein and is expressed with true late kinetics. To explore the function of ORF33 in viral lytic phase, we used site-directed mutagenesis and generated an ORF33-null mutant, taking advantage of the MHV-68 BAC system. We showed that the ORF33-null mutant is capable of viral DNA replication, early and late gene expression, capsid assembly, and DNA packaging, but incapable of virion release. The defect of ORF33-null mutant can be rescued in trans by an ORF33 expression plasmid.     

Murine gammaherpesvirus 68 open reading frame 45 plays an essential role during the immediate-early phase of viral replication

Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.     

Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.     

Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Murine gammaherpesvirus 68 lacking gp150 shows defective virion release but establishes normal latency in vivo

All gammaherpesviruses encode a virion glycoprotein positionally homologous to Epstein-Barr virus gp350. These glycoproteins are thought to be involved in cell binding, but little is known of the roles they might play in the whole viral replication cycle. We have analyzed the contribution of murine gammaherpesvirus 68 (MHV-68) gp150 to viral propagation in vitro and host colonization in vivo. MHV-68 lacking gp150 was viable and showed normal binding to fibroblasts and normal single-cycle lytic replication. Its capacity to infect glycosaminoglycan (GAG)-deficient CHO-K1 cells and NS0 and RAW264.7 cells, which express only low levels of GAGs, was paradoxically increased. However, gp150-deficient MHV-68 spread poorly through fibroblast monolayers, with reduced cell-free infectivity, consistent with a deficit in virus release. Electron microscopy showed gp150-deficient virions clustered on infected-cell plasma membranes. MHV-68-infected cells showed reduced surface GAG expression, suggesting that gp150 prevented virions from rebinding to infected cells after release by making MHV-68 infection GAG dependent. Surprisingly, gp150-deficient viruses showed only a transient lag in lytic replication in vivo and established normal levels of latency. Cell-to-cell virus spread and the proliferation of latently infected cells, for which gp150 was dispensable, therefore appeared to be the major route of virus propagation in an infected host.     

Herpes simplex virus glycoproteins gD and gE/gI serve essential but redundant functions during acquisition of the virion envelope in the cytoplasm

The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.     

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 RTA reactivates murine gammaherpesvirus 68 from latency

Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.     

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component

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.     

A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread

Viruses lack self-propulsion. To move in multi-cellular hosts they must therefore manipulate infected cells. Herpesviruses provide an archetype for many aspects of host manipulation, but only for alpha-herpesviruses in is there much information about they move. Other herpesviruses are not necessarily the same. Here we show that Murine gamma-herpesvirus-68 (MHV-68) induces the outgrowth of long, branched plasma membrane fronds to create an intercellular network for virion traffic. The fronds were actin-based and RhoA-dependent. Time-lapse imaging showed that the infected cell surface became highly motile and that virions moved on the fronds. This plasma membrane remodelling was driven by the cytoplasmic tail of gp48, a MHV-68 glycoprotein previously implicated in intercellular viral spread. The MHV-68 ORF58 was also required, but its role was simply transporting gp48 to the plasma membrane, since a gp48 mutant exported without ORF58 did not require ORF58 to form membrane fronds either. Together, gp48/ORF58 were sufficient to induce fronds in transfected cells, as were the homologous BDLF2/BMRF2 of Epstein-Barr virus. Gp48/ORF58 therefore represents a conserved module by which gamma-herpesviruses rearrange cellular actin to increase intercellular contacts and thereby promote their spread.