Effects of Major Capsid Proteins,Capsid Assembly,and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

The U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU_L17/pU_L25 interaction. We found that pU_L17 and pU_L25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U_L19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU_L17 (i) coimmunoprecipitated with pU_L25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU_L25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU_L25 in the absence of the triplex protein VP23 (encoded by the U_L18 gene), (iv) required pU_L25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU_L25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU_L25 in infected cell nuclei required pU_L17, pU_L32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU_L15. The data suggest that VP23 or triplexes augment the pU_L17/pU_L25 interaction and that VP23 and VP5 induce conformational changes in pU_L17 and pU_L25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU_L17/pU_L25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23, 29, 47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U_L6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U_L18 gene product, VP23, and one copy of the U_L38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U_L26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11, 12, 19, 25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U_L15, U_L17, U_L28, U_L32, and U_L33 genes (2, 4, 10, 18, 26, 28, 35, 46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U_L25 and U_L17 open reading frames (1, 17, 20). These proteins are located on the external surface of the viral capsid (24, 36, 44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU_L25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) associate with all capsid types, and pU_L17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28, 36, 37). How the U_L17 and U_L25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU_L17 and pU_L25 become incorporated into the capsid during the assembly pathway. Less pU_L25 associates with pU_L17(−) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU_L25 associates with capsids lacking DNA (30, 36).Both pU_L25 and pU_L17 are necessary for proper nucleocapsid assembly, but their respective deletion generates different phenotypes. Deletion of pU_L17 precludes DNA packaging and induces capsid aggregation in the nuclei of infected cells, suggesting a critical early function (28, 34), whereas deletion of pU_L25 precludes correct cleavage or retention of full-length cleaved DNA within the capsid (8, 20, 32), thus suggesting a critical function later in the assembly pathway.The current studies were undertaken to determine how pU_L17 and pU_L25 associate with capsids by studying their interaction and localization in the presence and absence of other capsid proteins.     

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the U_L6-encoded portal protein pU_L6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of U_L6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking U_L6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation''s detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pU_L6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.Immature herpesvirus capsids or procapsids consist of two shells: an inner shell, or scaffold, and an outer shell that is roughly spherical and largely composed of the major capsid protein VP5 (24, 38).The capsid scaffold consists of a mixture of the U_L26.5 and U_L26 gene products, with the U_L26.5 gene product (pU_L26.5, ICP35, or VP22a) being the most abundant (1, 12, 20, 21, 32, 38). The U_L26.5 open reading frame shares its coding frame and C terminus with the U_L26 gene but initiates at codon 307 of U_L26 (17). The extreme C termini of both VP22a and the UL26-encoded protein (pU_L26) interact with the N terminus of VP5 (7, 14, 26, 40, 41). Capsid assembly likely initiates when the portal binds VP5/VP22a and/or VP5/pU_L26 complexes (22, 25). The addition of more of these complexes to growing capsid shells eventually produces a closed sphere bearing a single portal. pU_L26 within the scaffold contains a protease that cleaves itself between amino acids 247 and 248, separating pU_L26 into an N-terminal protease domain called VP24 and a C-terminal domain termed VP21 (4, 5, 8, 9, 28, 42). The protease also cleaves 25 amino acids from pU_L26 and VP22a to release VP5 (5, 8, 9). VP21 and VP22a are replaced with DNA when the DNA is packaged (12, 29).When capsids undergo maturation, the outer protein shell angularizes to become icosahedral (13). One fivefold-symmetrical vertex in the angularized outer capsid shell is biochemically distinct from the other 11 and is called the portal vertex because it serves as the channel through which DNA is inserted as it is packaged (23). In herpes simplex virus (HSV), the portal vertex is composed of 12 copies of the portal protein encoded by U_L6 (2, 23, 39). We and others have shown that interactions between scaffold and portal proteins are critical for incorporation of the portal into the capsid (15, 33, 44, 45). Twelve amino acids of scaffold proteins are sufficient to interact with the portal protein, and tyrosine and proline resides within this domain are critical for the interaction with scaffold proteins and incorporation of the portal into capsids (45).One goal of the current study was to map domains and residues within the U_L6-encoded portal protein that mediate interaction with scaffold proteins. We show that the portal-scaffold interaction requires all but the first 18 and last 36 amino acids of pU_L6, as well as several tryptophan residues positioned throughout the portal protein.     

Phosphorylation of the UL31 Protein of Herpes Simplex Virus 1 by the US3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U_L31 and U_L34 gene products. While it is clear that the viral kinase encoded by the U_S3 gene regulates the localization of pU_L31/pU_L34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU_L31 is indispensable for the protein''s normal function and contains up to six serines that are phosphorylated by the U_S3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U_S3 kinase, changing the serines to alanine caused the pU_L31/pU_L34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U_S3 kinase-dead mutant. (iv) The replacement of the six serines of pU_L31 with glutamic acid largely restored the smooth distribution of pU_L34/pU_L31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U_S3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU_L31 by pU_S3 represents an important regulatory event in the virion egress pathway that can account for much of pU_S3''s role in nuclear egress. The data also suggest that the dynamics of pU_L31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.The U_L31 and U_L34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26, 27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26, 28). pU_L34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU_L31 and holds the latter in close approximation to the INM (16, 19, 26, 31, 36, 37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU_L31 and pU_L34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U_S3 protein is a promiscuous kinase that phosphorylates pU_L31, pU_L34, and several other viral and cellular components (1, 2, 5, 11, 15, 21-23, 25). In the absence of pU_S3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU_L31/pU_L34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12, 14, 27, 29), and (iii) the onset of infectious virus production is delayed (21, 29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U_S3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3, 4, 9, 10, 18, 30, 33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU_S3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU_S3 kinase responsible for the altered localization of the pU_L31/pU_L34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU_L34 was not responsible for the nuclear egress defects induced by the absence of pU_S3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU_S3-mediated phosphorylation of pU_L31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U_S3 kinase-dead phenotype, including the retention of virions in the perinuclear space, the mislocalization of the pU_L31/pU_L34 complex, and the delayed onset of virus replication, can be replicated by precluding pU_L31 phosphorylation in the presence or absence of pU_S3 kinase activity. The data also suggest that the dynamic phosphorylation of pU_L31 is important during the primary envelopment of nucleocapsids.     

The UL31 and UL34 Gene Products of Herpes Simplex Virus 1 Are Required for Optimal Localization of Viral Glycoproteins D and M to the Inner Nuclear Membranes of Infected Cells

U_L31 and U_L34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU_L31, pU_L34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U_L34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U_L34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U_L34 (P < 0.0001). pU_L34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU_L34/gD interaction in recruiting gD to the NM. The effects of U_L34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU_L31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U_L31 and U_L34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.Herpesvirus virions comprise a nucleocapsid containing genomic viral DNA, a proteinaceous tegument layer surrounding the nucleocapsid, and a virion envelope surrounding the tegument. The envelope of extracellular herpes simplex virus (HSV) virions contains glycoproteins gB, gC, gD, gE, gI, gG, gH, gK, gL, and gM (23, 51).As viewed by electron microscopy, nascent virions form as the nucleocapsid buds through densely staining regions of the nuclear membrane (NM) (21, 41). Electron tomograms of HSV perinuclear virions compared to those of extracellular virions infer that the former contain glycoproteins of considerably less glycosylation and a relatively sparse tegument layer compared to their counterparts in mature extracellular virions (6). The lower levels of glycosylation in HSV perinuclear virions are consistent with the fact that the lumen of the perinuclear space is continuous with that of the endoplasmic reticulum. Thus, the polysaccharide moieties of virion glycoproteins become fully processed as virions access Golgi enzymes during their egress to the extracellular space. Although the full proteome of the nascent perinuclear virion is unknown, immunogold studies have shown that they contain at least pU_L31, pU_L34, pUS3, gB, gC, gD, gH, gM, and the VP16 and pU_L11 tegument proteins in addition to the proteins that comprise the viral capsid (4, 5, 15, 25, 37, 40, 47, 50, 55).The U_L31 and U_L34 gene products of HSV-1 (pU_L31 and pU_L34, respectively) form a complex that localizes at the inner and outer NMs (INM and ONM, respectively) of infected cells (40). Both proteins are essential for nucleocapsid envelopment at the INM and become incorporated into nascent virions when nucleocapsids bud through the INM into the perinuclear space (39, 40, 42). The proteins and their essential role in nucleocapsid envelopment are conserved in all herpesvirus subfamilies (14, 20, 32, 45). pU_L31 of HSV-1 is a mostly hydrophobic phosphoprotein that is held in close approximation to the nucleoplasmic face of the INM by interaction with pU_L34, an integral membrane protein of type II orientation (33, 40, 46, 56). The first 248 amino acids of pU_L34 are predicted to reside in the nucleoplasm or cytoplasm, depending on whether the protein localizes in the INM or ONM, respectively. This is followed by an approximately 22-amino acid transmembrane domain with up to 5 amino acids residing in the perinuclear space or lumen of the endoplasmic reticulum.In the most prominent model of herpesvirion egress, the envelope of the perinuclear virion fuses with the ONM, releasing the deenveloped nucleocapsid into the cytoplasm, where it subsequently buds into cytoplasmic membranous organelles such as the Golgi or trans-Golgi network (34, 49). This model is supported by the observation that pU_L31 and pU_L34 are located in the perinuclear virion but not extracellular virions (18, 40). Thus, these proteins are lost from the virion upon fusion of the virion envelope with the ONM. Also supporting this egress model is the observation that deletion of both gB and gH causes virions to accumulate aberrantly in the perinuclear space (15). The involvement of gH and gB is potentially satisfying because these proteins comprise essential components of the machinery that mediates fusion of the virion envelope with the plasma or endosomal membranes during the initiation of infection (9, 12, 16, 44, 52). Moreover, expression of a combination of gB, gD, gH, and gL is sufficient to mediate fusion of cell membranes, whereas coexpression with gM or gK inhibits this fusion (3, 8, 11). Although the mechanism of fusion is unclear, gD is known to bind viral receptors on cell surfaces, and the structure of gB indicates features reminiscent of other viral fusion proteins (24, 35, 48). gD has been shown to interact with gB and gH at least transiently, suggesting that these interactions may be important for the fusion reaction (1, 2). Thus, fusion between the nascent and mature virion envelopes with target membranes may share mechanistic similarities.On the other hand, it is likely that the two fusion events are mechanistically distinct because (i) single deletion of either gH or gB precludes viral entry and cell/cell fusion but does not cause nascent virions to accumulate in the perinuclear space (9, 16, 31, 43) and (ii) the activity of a viral kinase encoded by U_S3 is dispensable for entry but believed to promote fusion of the perinuclear virion and ONM (28, 40). Moreover, the lack of glycoproteins from the pseudorabies virus perinuclear virion suggests that fusion is mediated by an entirely different mechanism in this system (26).The current study focuses on how glycoproteins are incorporated into the nascent virion. We show that optimal recruitment of gD to both leaflets of the NM and gM to the INM requires pU_L34 and pU_L31. We also show that immature gD interacts with pU_L34, suggesting a mechanism by which pU_L34 might recruit gD to the NM.     

Virion Incorporation of the Herpes Simplex Virus Type 1 Tegument Protein VP22 Occurs via Glycoprotein E-Specific Recruitment to the Late Secretory Pathway

The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE (ΔgEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.The herpesvirus tegument is the virion compartment located between the DNA-containing capsid and the virus envelope (6). Although it is well defined that the viral capsid assembles in the nucleus (37, 38) and the viral envelope is acquired from cellular membranes (3, 24), the mechanism of tegument protein acquisition is still to be established. At least 20 virus-encoded components are recruited into the herpes simplex virus type 1 (HSV-1) tegument (32), and there is increasing evidence to suggest that subsets of these proteins may be added as assembly progresses along the maturation pathway (28). To ensure efficient incorporation, it is likely that individual tegument proteins are specifically targeted to their cellular site of recruitment. Such targeting could involve interaction with a viral partner, a cellular partner, or both. A clearer understanding of how individual tegument proteins are acquired by newly assembling virions will help to define the herpesvirus assembly pathway.A number of protein-protein interactions between individual tegument proteins (13, 40, 42), and between tegument proteins and glycoproteins (19, 20, 22, 32), have been described that may provide useful insight into the assembly process. In particular, the interaction of tegument proteins with the cytoplasmic tails of virus glycoproteins provides an attractive mechanism for the virion recruitment of at least the outer components of the tegument. In the case of VP22, the homologues from pseudorabies virus (PRV) and HSV-1 have been shown to interact with the cytoplasmic tail of gE (19, 20, 32). However, the role of this interaction in virus infection has not yet been clearly defined and the fact that additional glycoprotein interactions have been described, with gM in the case of PRV and gD in the case of HSV-1, may point to potential redundancy in the mechanism of VP22 packaging (4, 19, 20). In addition, we and others have previously shown that HSV-1 VP22 interacts directly with a second tegument protein, namely, VP16 (13, 33), an interaction that could provide an alternative route for VP22 to enter the virion. In a previous study, we concluded that the region of VP22 containing its VP16 interaction domain was required but not sufficient for optimal VP22 packaging into the assembling virion, with an additional C-terminal determinant also involved (23). We also demonstrated that the same region of VP22 that was required for virion packaging was essential to target the protein to its characteristic cytoplasmic trafficking complexes, suggesting that these specific sites may be the location in the cell for VP22 assembly into the virion (23). Since that study, O''Regan and coworkers have reported that the C-terminal half of HSV-1 VP22 also contains the binding site for gE (32), providing a possible candidate for an additional VP22 binding partner. Furthermore, as HSV-1 VP22 has been shown to bind to gD and PRV VP22 interacts with gM, it is possible that the C terminus of VP22 contains a gD and/or a gM binding site (4, 20).In the present study, we aimed to clarify the molecular mechanism by which VP22 is recruited into the virus particle. We show that HSV-1 VP22 binds efficiently to VP16, gE, and gM in the infected cell, but we cannot detect an interaction with gD. We show that the packaging domain of VP22 binds to the cytoplasmic tail of gE but not gM and that the same region of VP22 is recruited to the secretory pathway by gE in the absence of other virus proteins. Finally, we show that a mutant VP22 protein lacking a 14-residue peptide from its packaging domain is unable to interact with gE during infection, exhibits a different subcellular localization, and fails to assemble into the virus particle. This is the first characterization of a single protein-protein interaction essential for the packaging of an HSV-1 tegument protein.     

Time-Dependent Transformation of the Herpesvirus Tegument

All herpesviruses have a layer of protein called the tegument that lies between the virion membrane and the capsid. The tegument consists of multiple, virus-encoded protein species that together can account for nearly half the total virus protein. To clarify the structure of the tegument and its attachment to the capsid, we used electron microscopy and protein analysis to examine the tegument of herpes simplex virus type 1 (HSV-1). Electron microscopic examination of intact virions revealed that whereas the tegument was asymmetrically distributed around the capsid in extracellular virions, it was symmetrically arranged in cell-associated virus. Examination of virions after treatment with nonionic detergent demonstrated that: (i) in extracellular virus the tegument was resistant to removal with Triton X-100 (TX-100), whereas it was lost nearly completely when cell-associated virus was treated in the same way; (ii) the tegument in TX-100-treated extracellular virions was asymmetrically distributed around the capsid as it is in unextracted virus; and (iii) in some images, tegument was seen to be linked to the capsid by short, regularly spaced connectors. Further analysis was carried out with extracellular virus harvested from cells at different times after infection. It was observed that while the amount of tegument present in virions was not affected by time of harvest, the amount remaining after TX-100 treatment increased markedly as the time of harvest was increased from 24 h to 64 h postinfection. The results support the view that HSV-1 virions undergo a time-dependent change in which the tegument is transformed from a state in which it is symmetrically organized around the capsid and extractable with TX-100 to a state where it is asymmetrically arranged and resistant to extraction.All herpesviruses have a tegument, a layer of protein located between the virus membrane and the capsid. Depending on the virus species, the tegument can be 20 to 40 nm in thickness, and it may be uniformly or asymmetrically distributed about the capsid (7, 17, 24, 33). The tegument is composed predominantly of virus-encoded proteins that together can account for up to half or more of the total virion protein mass. Tegument proteins are thought to be those involved in the early stages of infection before progeny virus proteins are synthesized.The tegument has been most thoroughly studied in herpes simplex virus type 1 (HSV-1). Examination of virions by electron microscopy has demonstrated that the tegument is not highly structured. Its morphology is described as predominantly granular with fibrous elements also present (7, 19). Analysis by cryo-electron microscopy, followed by icosahedral reconstruction has shown that the tegument is not icosahedrally ordered, although a small amount of tegument density is observed close to the capsid surface at the pentons (3, 47).The HSV-1 tegument is composed of approximately 20 distinct, virus-encoded protein species whose amounts vary considerably. The predominant components are UL47, UL48, and UL49, each of which occurs in more than 800 copies per virion (8, 46). In contrast, others, such as RL2 (ICP0), RS1 (ICP4), UL36, and UL37, occur in ∼100 copies or less. Trace amounts of host cell-encoded proteins are also present (15). Many of the tegument proteins are required for virus replication (34), and functions have been defined for most (9, 12, 31, 40).Biochemical studies have demonstrated that the tegument makes noncovalent contacts with both the virus capsid and the membrane. Studies of capsid-tegument contacts have emphasized binding of UL36, a tegument protein, to UL25, a capsid protein located near the vertices and involved in DNA encapsidation (5, 20, 29). Other tegument proteins such as UL48 (VP16), UL37, and UL49 (VP22) are found to associate with UL36 and may be bound to the capsid indirectly by way of UL36 (13, 44). UL16 binds reversibly to the capsid while UL46 (VP11/12) has been shown to bind to both the membrane and the capsid (21, 22, 26). Binding of tegument proteins to the membrane has been shown to occur by way of attachment to UL11 (45) and also to the internal domains of membrane glycoproteins, including glycoprotein D (gD), gH, and gE (4, 6, 11).We describe here the results of a study in which electron microscopy and protein analysis were used to clarify the structure of the HSV-1 tegument and its attachment to the capsid. The study was designed to extend the observation that most of the HSV-1 tegument remains attached to the capsid when the membrane is removed from the virus by treatment with nonionic detergent (19). Cell-associated and extracellular virions were compared after treatment with Triton X-100 (TX-100).     

Structure and Capsid Association of the Herpesvirus Large Tegument Protein UL36

The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (∼50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.All herpesviruses have a tegument, a layer of protein located between the virus capsid and membrane. The tegument accounts for a substantial proportion of the overall virus structure. Its thickness (30 to 50 nm), for example, may be comparable to the capsid radius, and tegument proteins can account for 40% or more of the total virion protein. Herpesvirus tegument proteins are thought to function promptly after initiation of infection, before expression of virus genes can take place (11, 13, 14, 21, 33, 37).Electron microscopic analysis of virions has demonstrated that the tegument is not highly structured (9, 22). It does not have icosahedral symmetry like the capsid, and it may be uniformly or asymmetrically arranged around the capsid (26). Tegument structure is described as fibrous or granular, and its morphology is found to change as the virus matures. Studies with herpes simplex virus type 1 (HSV-1), for example, indicate that the tegument structure is altered in cell-associated compared to extracellular virus (26).The tegument has been most thoroughly studied in HSV-1, where biochemical analyses indicate that it is composed of approximately 20 distinct, virus-encoded protein species. The predominant components are the products of the genes UL47, UL48, and UL49, with each protein present in 800 or more copies per virion (12, 40). Other tegument proteins can occur in 100 or fewer copies, and trace amounts of cell-encoded proteins are also present (17). Tegument proteins are classified as inner or outer components based on their association with the capsid after it enters the host cell cytoplasm. The inner tegument proteins (UL36, UL37, and US3) are those that remain bound to the capsid after entry, while the others (the outer tegument proteins) become detached (7, 18).The HSV-1 UL36 protein has the potential to play a central role in organizing the overall structure of the tegument. With a length of 3,164 amino acids, UL36 could span the thickness of the tegument multiple times. One hundred to 150 UL36 molecules are present in the tegument (12), and they are bound to the capsid by way of an essential C-terminal domain (2, 16). UL36 is able to bind the major tegument components by way of documented direct (UL37 and UL48) and indirect (UL46, UL47, and UL49) contacts (6, 15, 24, 38).Here we describe the results of studies designed to test the idea that UL36 serves to organize the tegument structure. Beginning with infectious virus, a novel method has been used to isolate capsids that contain UL36 and UL37 but lack the virus membrane and are depleted of all other tegument proteins. These capsids (T36 capsids) were examined by electron microscopy to clarify the structure of UL36 and UL37 molecules and their location on the capsid surface.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.The herpesvirus virion is composed of an icosahedral nucleocapsid containing the viral genome, an envelope of cellular origin with inserted viral (glyco)proteins, and a tegument which links nucleocapsid and envelope comparable to the matrix of RNA viruses. The herpesvirus tegument contains a multitude of viral and cellular proteins (reviewed in references 45 and 46). Tegument proteins execute various regulatory and structural functions, including activation of viral gene expression (2), modulation of the host cell for virus replication (26, 51, 55), and mediation of posttranslational modification of proteins (10, 27, 50). Numerous interactions have been identified among tegument proteins, between tegument and capsid proteins, and between tegument and envelope proteins (7, 14, 16, 18, 33, 36, 42, 53, 58-61).The largest tegument proteins found in the herpesviruses are homologs of pUL36 of herpes simplex virus type 1 (HSV-1). Pseudorabies virus (PrV) pUL36 consists of 3,084 amino acids (aa) with a molecular mass of 324 kDa (33). PrV and HSV-1 pUL36 are essential for viral replication (13, 15). In their absence, nonenveloped nucleocapsids accumulate in the cytoplasm. Whereas in several studies nuclear stages like cleavage and packaging of the viral DNA as well as nuclear egress were not found affected (13, 15), another study indicated an effect of pUL36 deletion on PrV nuclear egress (41).pUL36 homologs complex with another tegument protein, pUL37, as has been shown for HSV-1 (59), PrV (15, 33), and human cytomegalovirus (3, 23), and the interacting region on pUL36 has been delineated for PrV (33) and identified at the amino acid level for HSV-1 (47). Deletion of the pUL37 interaction domain from PrV pUL36 impedes virion formation in the cytosol but does not block it completely, yielding a phenotype similar to that of a pUL37 deletion mutant (31). This indicates an important but nonessential role for pUL37 and the pUL37 interaction domain in pUL36 in virion formation (15). In contrast, absence of pUL37 completely blocks virion formation in HSV-1 (11, 38).pUL36 is stably attached to the nucleocapsid (39, 43, 56), remains associated with incoming particles during transport along microtubules to the nuclear pore (21, 40, 52), and is required for intracellular nucleocapsid transport during egress (41). In contrast, absence of pUL37 delays nuclear translocation of incoming PrV nucleocapsids but does not abolish it (35). HSV-1 pUL36 is involved not only in transport but also in docking of nucleocapsids to the nuclear pore (9), and proteolytic cleavage of pUL36 appears to be necessary for release of HSV-1 DNA into the nucleus (24).Immunoelectron microscopical studies of PrV-infected cells showed that pUL36 is added to nucleocapsids prior to the addition of pUL37 (33). Since neither pUL36 nor pUL37 was detected on primary enveloped PrV virions, it was concluded that acquisition of tegument takes place in the cytoplasm (20). However, conflicting data exist whether pUL36 is present in the nucleus, and whether it is already added onto the capsids in this cellular compartment. Indirect immunofluorescence, immunoelectron microscopy and mass spectrometry of intranuclear capsids yielded discrepant results. By immunofluorescence HSV-1 pUL36 was detected both in the cytoplasm and in the nucleus (1, 42, 48). However, whereas one study detected the protein on nuclear C-capsids by Western blotting (6), it was not found by cryo-electron microscopy and mass spectrometry (57). In contrast, the C terminus of PrV pUL36 was suggested to direct pUL36 to capsid assemblons in the nucleus (37) by binding to capsid-associated pUL25 (8), although pUL36 could not be detected in the nucleus during PrV infection (33). These differing results in HSV-1 and between HSV-1 and PrV might be due to the fact that pUL36 could be processed during the replication cycle and that the resulting subdomains may exhibit selective localization patterns (24, 28).Amino acid sequence analyses of HSV-1 and PrV pUL36 revealed several putative nuclear localization signals (NLS) (1, 4, 5, 49). HSV-1 pUL36 contains four of these NLS motifs (49). Functionality in nuclear localization of a reporter protein was shown for the NLS motif at aa 425 (1). This motif is highly conserved in herpesvirus pUL36 homologs pointing to an important function (1). Besides this conserved NLS (designated in this report as NLS1), two other NLS motifs are predicted in PrV pUL36. One is located adjacent to NLS1 (aa 288 to 296) at aa 315 to 321 (NLS2), and a third putative NLS motif is present in the C-terminal half of the protein (aa 1679 to 1682; NLS3) (4). Whereas this may be indicative for a role for pUL36 inside the nucleus, NLS motifs might also be involved in transport to the nucleus along microtubules (54) and docking at the nuclear pore complex (49).The discrepancy in pUL36 localization and the putative presence of pUL36 cleavage products with specialized functions and localization prompted us to generate monospecific antisera covering the major part of PrV pUL36 and to study localization of PrV pUL36 by immunofluorescence during viral replication and after transient transfection and by immunoelectron microscopy of infected cells.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.In recent years, virus-like particles (VLPs), which are formed upon recombinant expression of the viral matrix and/or surface glycoproteins, have been recognized as representing powerful tools for developing novel vaccines and investigating certain aspects of the viral replication cycle (24, 44, 59, 63). Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16, 38, 42, 54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase. Expression of dominant-negative (DN) VPS4 mutants, which lack the ability to bind or hydrolyze ATP, blocks recycling of the ESCRTs and induces the formation of enlarged endosomes lacking internal vesicle accumulation (2, 3, 7). The inward budding of vesicles into the MVBs is topologically similar to the budding of viruses, since the vesicles bud away from the cytosol and into the lumen (reviewed in references 1, 20, and 26). Therefore, it is not entirely surprising that viruses use the cellular ESCRT machinery to organize the budding of viral progeny. Interactions between viral matrix proteins and ESCRTs occur through tetrapeptide motifs, known as late domains, which were first identified in retroviruses. Known late domains consist of the amino acid sequence P(T/S)AP, PPxY, or YxxL, where “x” represents any amino acid (19, 25, 62). The P(T/S)AP motif, for example, mediates interaction with tumor susceptibility gene 101 (Tsg101) (16, 36, 57); the PPxY motif mediates binding to WW domains of Nedd4-like ubiquitin ligases (9, 22); and the YxxL motif mediates interaction with AIP1/Alix (35, 47, 58). Recently, a novel late-domain motif, FPIV, has been identified in paramyxoviruses (46), and it is thought that additional late-domain motifs remain to be discovered (for a review, see reference 5).Inhibition of the VPS pathway has been shown to inhibit the budding of various viruses that are released with the help of ESCRTs. However, the budding of viruses and VLPs depends on the activity of ESCRTs to different degrees. Downregulation of Tsg101, a member of the ESCRT-I complex, inhibited the release of VLPs mediated by lymphocytic choriomeningitis virus Z protein and Marburg virus (MARV) VP40 (42, 54) but did not substantially inhibit the release of Gag-induced VLPs of Moloney murine leukemia virus and Rous sarcoma virus or that of matrix protein-induced VLPs of rabies virus (16, 27, 38). Expression of DN VPS4 inhibited the release of VLPs induced by the Gag proteins of Rous sarcoma virus and Moloney murine leukemia virus (16, 38) as well as that of VLPs induced by Lassa virus Z protein (55) but had no effect on the budding of rabies virus and cytomegalovirus (13, 27). These data indicate that in spite of the presence of late-domain motifs, a block in the VPS pathway may not always be critical for the budding of VLPs. In addition, the lack of known late domains in many enveloped viruses raises the question of whether they use other entry points into the VPS pathway or whether they exploit entirely different mechanisms of budding (60). To date, knowledge of how viral matrix proteins engage cellular machineries, such as the VPS pathway, to induce viral budding at the plasma membrane is very limited (8).The matrix protein VP40 of MARV contains only one known late-domain motif, PPPY, and a recent study showed that mutation of this late domain inhibited the release of VP40-induced VLPs. In addition, depletion of Tsg101 reduced the release of VP40-induced VLPs, suggesting that ESCRT-I is involved in this process (54). Whether a functional VPS pathway is important for the release of MARV VP40-induced VLPs or MARV particles remains unknown.VLPs induced by many viral matrix proteins have a morphology similar to that of cellular vesicles, which makes it difficult to separate the spherical VLPs from released cellular vesicles (4, 17, 53). In contrast, VLPs induced by the filovirus matrix protein VP40 are elongated and similar in morphology to viral particles (30, 49). Nevertheless, we observed that the supernatants of cells expressing VP40 contained various populations of particles with different morphologies. This raised the questions of whether the different particles are released by the same mechanism, whether they are all induced by VP40, and whether they are dependent on the same cellular pathways.The aim of the present study was to analyze the populations of particles released from cells expressing the MARV matrix protein VP40 and to gain further insights into the interaction between MARV and the cellular machinery involved in the budding of VLPs and MARV particles.We found that cells expressing VP40 released vesicular and filamentous particles, which could be separated by gradient centrifugation. Fractions with mainly vesicular particles represented a mixture of vesicles containing exclusively cellular proteins and vesicles also containing VP40 and few short filamentous particles. Longer filamentous particles, whose morphology resembled that of MARV particles but which displayed a much higher variability in length (400 nm to 5 μm), were found in denser gradient fractions. Filamentous VP40-induced VLPs were able to sort out cellular proteins efficiently. Release of VP40-induced filamentous VLPs was supported by the late-domain motif present in VP40, and inhibition of the cellular ESCRT machinery reduced the amount of these VLPs in the supernatant. Interestingly, the release of VLPs induced by a mutant of VP40 lacking the late domain was also reduced by inhibition of the cellular ESCRT machinery. Expression of a DN mutant of VPS4 diminished the budding of infectious MARV particles by 50%, a finding consistent with the idea that the activity of the ESCRT machinery supports viral budding but is not essential.     

Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner,pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation

Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages.Murine cytomegalovirus (MCMV) US22 family genes M36, M139, M140, and M141 promote efficient replication of the virus in macrophages (1, 8, 12, 17). The M139, M140, and M141 genes are clustered within the MCMV genome and appear to function cooperatively (10, 12). During infection, the protein M140 (pM140) forms a stable complex with pM141, and one or more larger complexes are formed by the addition of M139 gene products (15). Although these complexes are evident in infected fibroblasts as well as macrophages, they are required for optimal MCMV replication selectively in macrophages (1, 17). In the absence of M140, virion assembly in macrophages is defective, likely due to the reduced levels of the major capsid protein and tegument protein M25 (11). pM140 also confers stability to its binding partner, pM141; in the absence of the M140 gene, the half-life of pM141 is reduced from 2 h to 1 h (12). Deletion of M141 compromises virus replication in macrophages (12), and pM141 directs pM140 to a perinuclear region of infected macrophages adjacent to an enlarged microtubule organizing center with characteristics of an aggresome (11, 15). Aggresomes are sites where proteins are targeted for degradation by either the proteasome or autophagy (3, 6, 19). We therefore hypothesized that complexing of pM141 to pM140 rescues pM141 from degradation by the proteasome and/or autophagy.     

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.Human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]), the prototypical betaherpesvirus, is ubiquitous in humans and establishes a persistent infection in the host (19). HCMV also reinfects healthy seropositive individuals, suggesting another mechanism for maintaining persistence in a population (9). Intrauterine transmission and HCMV infection of the developing fetus constitute a leading viral cause of birth defects (32). HCMV is also a leading cause of opportunistic infections in immunocompromised patients, including transplant recipients and patients with AIDS (10, 20). HCMV infection has also been implicated as a cofactor in such diverse diseases as atherosclerosis and cancer (8, 17, 33, 66).HCMV replicates its genome in the nucleus, and acquisition of the final tegument and envelope is thought to occur in the cytoplasm of infected cells (73, 77). Envelopment of HCMV has been reported to occur by budding into cytoplasmic vacuoles that are composed of HCMV glycoproteins required for the assembly of infectious virions (37). The fully mature virus is released from the cell through either exocytosis or, possibly, lysis of the infected cells (56). The nucleic acid-containing capsid is embedded in a proteinaceous tegument layer that occupies the space between the nucleocapsid and the envelope. The tegument contains approximately 40% of the virion protein mass and approximately 20 to 25 known virion proteins, most of which are phosphorylated (40, 44). The assembly pathway and protein interactions required for formation of the tegument layer and the role of individual tegument proteins in the replication and assembly of infectious HCMV remain poorly understood. Deletion of viral genes encoding some tegument proteins results in varying levels of impairment in virus production (11-13, 35, 43, 45, 53, 68). Some tegument proteins, such as pp28 (pUL99) and ppUL25, are expressed only in the cytoplasm of infected cells during HCMV replication, whereas others, such as ppUL53 and pp65 (pUL83), are expressed in the nuclei of cells early in infection but are localized predominantly in the cytoplasm late in infection (68). Others, such as the tegument protein ppUL69, are expressed only in the nuclei of infected cells. Finally, the intracellular localization of other tegument proteins, such as pp150 (pUL32), is less well defined in that both nuclear and cytoplasmic localizations have been described (34, 68).HCMV pp150 (basic phosphoprotein [BPP], pUL32) is the 1,048-amino-acid product of the UL32 gene of HCMV and an abundant constituent of the HCMV virion. Homologues of pp150 are found in other betaherpesviruses, including chimpanzee CMV, rat CMV, mouse CMV, HHV-6, and HHV-7, but not in alpha- or gammaherpesviruses (2). It is expressed late in HCMV infection (15, 68). It comprises 9.1% of infectious virion mass and 2% of the mass of dense bodies, suggesting that it is preferentially incorporated into virions (87). It has an estimated molecular mass of 113 kDa and is posttranslationally modified by phosphorylation and glycosylation, resulting in a molecular mass of 150 kDa in purified virus preparations analyzed by SDS-PAGE (41, 42, 65). pp150 has been classified as a tegument protein based on its presence in virion preparation, noninfectious enveloped particles, and cytoplasmic nucleocapsids but not in immature nuclear capsids (27, 28, 40). It has been suggested that pp150 contacts the capsids through the distal end of the capsomeres or through the triplex subunits that interlink them (16, 86). It has been reported to bind HCMV capsids in vitro through its amino one-third (6). We have also noted association of pp150 with the virion capsid by cryo-immunoelectron microscopy (W. Britt and H. Zhou, UCLA, Los Angeles, CA, unpublished findings). In primary human foreskin fibroblast (HFF) cells infected with HCMV, pp150 accumulates in a juxtanuclear structure that is termed the assembly compartment (AC), which colocalizes with markers of the distal secretory pathway and with other tegument proteins, including pp28 and pp65 and envelope glycoproteins gB, gH, and gM/gN (68). The virus-induced AC appears to overlap with microtubules emanating from the microtubule-organizing center (MTOC) and is proposed to be a cytoplasmic site of virion assembly (37, 68).The function of pp150 is unknown, although its close association with the nucleocapsid suggests potential involvement in nuclear targeting during entry and in nuclear targeting of the encapsidated viral DNA, capsid tegumentation, and/or envelopment late in infection. It is essential for production of infectious virus, since the deletion of the UL32 open reading frame (ORF) leads to loss of virus replication and has been reported to be important in cytoplasmic maturation of HCMV, especially in viral egress (2, 22, 84, 91, 92). In cells infected with ΔUL32 virus, which lacks pp150, fewer virus particles accumulated in the cytoplasm, although nuclear steps in virus assembly were not affected (84). It was also observed that in the absence of pp150, nucleocapsids were present in the viral assembly compartment but failed to proceed further to vesicle transport-associated release (84). These observations, together with pp150 abundance in the virion, suggest a primary contribution for this structural protein in the morphogenesis and/or cytoplasmic transport of progeny virion particles to sites of virion envelopment.Since pp150 has no predicted intracellular trafficking signals, its localization to the AC in virus-infected cells has been postulated to be dependent on interactions with cellular and/or viral proteins. Using yeast two-hybrid (Y2H) screening experiments we identified the cellular protein Bicaudal D1 (BicD1) as an interacting cellular protein. Bicaudal D was originally defined as a Drosophila protein that is involved in establishing the asymmetric cytoplasm in the developing oocyte (82, 89). Two homologues of Bicaudal D, BicD1 and BicD2, have been reported in humans, and these proteins have been reported to be involved in dynein-mediated microtubule transport as well as in COPI-independent Golgi-endoplasmic reticulum (ER) transport (38, 39, 55). Microtubule-dependent transport is an energy-dependent active transport system that includes both positive-end (directed away from the MTOC) and negative-end (directed toward the MTOC) transport. The direction of transport depends on cargo interactions with the molecular motors directing this transport, with dynein being associated with negative-end transport and kinesin with positive-end transport. BicD1 colocalizes with Rab6a in the trans-Golgi network and on cytoplasmic vesicles that associate with Golgi membranes in a Rab6-dependent manner secondary to a Rab6 binding domain at the C terminus of BicD1, suggesting an important role for BicD1 as an adaptor for dynein-dependent transport in the cell (55). In addition to having a role in the Golgi-ER trafficking, BicD1 has been shown to regulate anchoring of microtubules to the centrosome, as BICD1/2 knockdown induced microtubule unfocusing, with microtubules no longer appearing to radiate from the centrosome (26). BicD1 binds to its cargo via its C-terminal domain and to the dynein motor via its N-terminal domain (38). In this study we demonstrated that pp150 and BicD1 interact and that this interaction was required for localization of pp150 to the AC in virus-infected cells. In addition, we demonstrated that inhibition of BicD1 expression by short hairpin RNA (shRNA) led to a reduction in the yield of infectious virus. Finally, we demonstrated that formation of the AC and the assembly of infectious virions were dynein dependent, suggesting a critical role in microtubules in the production of infectious HCMV. Together, these results argue that HCMV replication is dependent on efficient localization of pp150 to the AC through its interaction with BicD1 and that pp150 localization to the AC is dynein dependent.     

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.