The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.All members of the Herpesviridae encode a conserved alkaline DNase that displays limited homology to bacteriophage λ red α (2, 24), an exonuclease that acts in conjunction with the synaptase red β to catalyze homologous recombination between DNA molecules (23). The most thoroughly characterized member of the herpesvirus alkaline nuclease family is encoded by the herpes simplex virus type 1 (HSV-1) gene UL12 (7, 9, 22). HSV-1 UL12 has both endo- and exonuclease activity (15-17, 36) and binds the viral single-stranded DNA binding protein ICP8 (37, 39) to form a recombinase that displays in vitro strand exchange activity similar to that for red α/β (27). UL12 localizes to the nucleus (26) where it plays an important, but as-of-yet ill-defined, role in promoting the production of mature packaged unit-length linear progeny viral DNA molecules (12, 20), perhaps via a recombination mechanism (27, 28). The importance of UL12 is documented by the observation that UL12 null mutants display a ca. 1,000-fold reduction in the production of infectious progeny virions (41).HSV-1 also produces an amino-terminally truncated UL12-related protein termed UL12.5, which is specified by a separately promoted mRNA that initiates within UL12 coding sequences (7, 9, 21). UL12.5 is translated in the same reading frame as UL12 but initiates at UL12 codon 127 and therefore lacks the first 126 amino acid residues of the full-length protein. UL12.5 retains the nuclease and ICP8 binding activities of UL12 (4, 14, 26) but does not accumulate to high levels in the nucleus (26) and is unable to efficiently substitute for UL12 in promoting viral genome maturation (14, 21). We recently showed that UL12.5 localizes predominantly to mitochondria, where it triggers massive degradation of the host mitochondrial genome early during HSV infection (31). Mammalian mitochondrial DNA (mt DNA) is a 16.5-kb double-stranded circle located within the mitochondrial matrix that encodes 13 proteins involved in oxidative phosphorylation and the RNA components of the mitochondrial translational apparatus (reviewed in reference 10). Inherited mutations that inactivate or deplete mt DNA impair oxidative phosphorylation, leading to a wide range of pathological conditions, including neuropathy and myopathy (reviewed in references 8 and 40). Thus, although the contribution of mt DNA depletion to the biology of HSV infection has yet to be determined, it likely has a major negative impact on host cell functions.UL12 and UL12.5 provide a striking example of a pair of highly related proteins that share a common biochemical activity yet differ markedly in subcellular location and biological function. The basis for their distinct subcellular localization patterns is of considerable interest, as the only difference in the primary sequences is that UL12.5 lacks the first 126 residues of UL12. Reuven et al. (26) demonstrated that this UL12-specific region contains one or more signals able to target enhanced green fluorescent protein (eGFP) to the nucleus. However, the determinants of the mitochondrial localization of UL12.5 have not been previously examined. Most proteins that are imported into the mitochondrial matrix bear a matrix targeting sequence that is located at or close to the amino terminus (reviewed in references 25 and 38). We speculated that UL12.5 bears such an amino-terminal matrix targeting sequence and that the function of this element is masked in the full-length UL12 protein by the UL12-specific amino-terminal extension, which contains the nuclear localization signal(s) (NLS). Our results broadly support this hypothesis and indicate that the mitochondrial localization sequence of UL12.5 is located ca. 60 residues from its N terminus.     

Mutational Analysis of the Herpes Simplex Virus Type 1 DNA Packaging Protein UL33

The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging.During the packaging of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1), the cleavage of replicated concatemeric viral DNA into single-genome lengths is tightly coupled to its insertion into preassembled spherical procapsids. Upon genome insertion, the internal scaffold protein of the procapsid is lost, and the capsid shell angularizes. Genetic analysis has revealed that successful packaging requires a cis-acting DNA sequence (the a sequence) together with seven proteins, encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes (6, 10). By analogy with double-stranded bacteriophage, the encapsidation of HSV-1 DNA is thought to be mediated by a heteromultimeric terminase enzyme. It is envisaged that the terminase is involved in the recognition of packaging signals present in the concatemers and the association with procapsids via an interaction with the capsid portal protein. Terminase initiates packaging by cleaving at an a sequence present between adjacent genomes within concatemers and subsequently provides energy for genome insertion through the hydrolysis of ATP. Packaging is terminated by a second cleavage event at the next similarly orientated a sequence, resulting in the encapsidation of a unit-length genome.An accumulating body of evidence suggests that the HSV-1 terminase is comprised of the UL15, UL28, and UL33 gene products. Viruses lacking a functional version of any of these three proteins are unable to initiate DNA packaging, and uncleaved concatemers and abortive B-capsids (angularized forms containing scaffold but no DNA) accumulate in the nuclei of infected cells (2, 4, 5, 11, 25, 27, 30, 36, 38). Protein sequence comparisons revealed a distant relationship between UL15 and the large subunit of bacteriophage T4 terminase, gp17, including the presence of Walker A and B box motifs characteristic of ATP binding proteins (13). Subsequent experiments demonstrated that point mutations affecting several of the most highly conserved residues abolished the ability of the resulting mutant viruses to cleave and package viral DNA (26, 39). The UL28 component has been reported to interact with the viral DNA packaging signal (3), a property shared with the homologous protein of human cytomegalovirus (CMV), UL56 (9). Furthermore, both UL15 and UL28 are able to interact with UL6 (33, 37), which form a dodecameric portal complex through which DNA is inserted into the capsid (22, 23, 31). Within the terminase complex, strong interactions have previously been reported between UL15 and UL28 and between UL28 and UL33 (1, 7, 17, 19, 34). Evidence also suggests that UL15 and UL33 may be able to interact directly, albeit more weakly than UL28 and UL33 (7, 15). Temperature-sensitive (ts) lesions in UL33 or UL15 reduced both the interaction of the thermolabile protein with the other members of the terminase complex and viral growth at the nonpermissive temperature (36). Recent evidence suggests that the terminase complex assembles in the cytoplasm and is imported into the nucleus via a mechanism involving a nuclear localization signal within UL15 (35). UL15 is also necessary for the localization of the terminase to nuclear sites of DNA replication and packaging (15). At present, the enzymatic activities necessary for DNA packaging have not been demonstrated for either the complex or individual subunits of the HSV-1 terminase.This study concerns the UL33 protein, which, at 130 residues, is the smallest subunit of the presumptive terminase (7, 27). No specific role in terminase activity has yet been ascribed to UL33, but several possibilities have been proposed including (i) ensuring correct folding or assembly of the complex, (ii) regulating the functions of the other subunits, (iii) performing an essential enzymatic role per se, and (iv) ensuring correct localization of the terminase to sites of DNA packaging (7). However, recent immunofluorescence studies using mutants with defects in the individual terminase subunits suggest that UL33 is unlikely to be involved in this last function (15).In order to further investigate the role of UL33 in the cleavage-packaging process, we utilized transposon-mediated mutagenesis to introduce insertions of five codons throughout the UL33 ORF. We report the generation and characterization of 15 mutants in terms of their ability to support viral growth and DNA packaging and to interact with the terminase component UL28.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2, 7, 34, 36, 39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11, 13, 15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm and transiently localizes to nucleoli (23). In the absence of other viral proteins, UL24 accumulates in the Golgi apparatus and in the nucleus, where it usually exhibits a diffuse staining pattern, but in a minority of cells it is detected in nucleoli (3).During infection, the formation of the viral replication compartments in the nucleus and the action of several viral proteins result in a remodeling of the nucleus. Chromatin is marginalized (29, 40), promyelocytic leukemia bodies are dispersed (26, 27), and the nuclear lamina is disrupted (33, 37). HSV-1 infection also affects the nucleolus, a prominent nuclear substructure implicated in the synthesis of rRNA, cell cycle regulation, and nucleocytoplasmic shuttling (5). Nucleoli become elongated following infection, and the synthesis of mature rRNA is reduced (4, 38, 42). Several HSV-1 proteins have been shown to localize to, or associate with, the nucleolus (12). The viral protein VP22 associates with the nucleolus and with dispersed nucleolin in HSV-1-infected cells (22), and RL1, US11, and ICP0 have also been shown to localize to nucleoli (24, 30, 35). Previously we showed that nucleolin is dispersed throughout the nucleus upon HSV-1 infection and that UL24 is involved in this nuclear modification (23). We further found that the N-terminal portion of UL24 is sufficient to induce the redistribution of nucleolin in the absence of other viral proteins (3).In this study, we sought to test the hypothesis that the endonuclease motif, which is made up of some of the most highly conserved residues in UL24, is important for the dispersal of nucleolin. A panel of substitution mutations in UL24 was generated, and the impact on the function of UL24 was assessed.     

The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

Kaposi''s sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi''s sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 Å. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8''s mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.Most if not all organisms with DNA genomes have mechanisms to ensure processive DNA synthesis. In bacteria, archaea, and eukaryotes, DNA polymerase subunits include a catalytic subunit and a processivity factor, often referred to as a “sliding clamp.” In these organisms, a clamp loader protein is required to assemble the processivity factor onto the DNA (27, 37). The bacterial sliding (beta) clamp is made up of homodimers of a subunit that comprises three structurally similar subdomains (26), whereas archaeal and eukaryotic proliferating cell nuclear antigen (PCNA) is composed of homotrimers that comprise two structurally similar subdomains (27, 37). For both of these clamps, the monomers assemble head-to-tail to form a closed homodimeric or homotrimeric ring, respectively, around the DNA. In these organisms, a clamp loader protein is required to efficiently load the clamp onto DNA, using an ATP-dependent process. Once loaded on DNA, the processivity factor is capable of binding directly to the DNA polymerase, conferring extended strand synthesis without falling off of the template (50).Herpesviruses encode their own DNA polymerases. However, unlike bacteria, archaea, and eukaryotes, herpesviruses do not encode clamp loaders to assemble their processivity factors onto the DNA. Yet, the accessory subunits of the herpesvirus DNA polymerases still associate with DNA with nanomolar affinity to enable long-chain DNA synthesis (9, 16, 23, 25, 29, 35, 44, 46, 53, 56). Human herpesviruses are divided into three classes, namely, the alpha-, beta-, and gammaherpesviruses, based on homologies found in their genomic organization as well as in protein sequences and function (45). Crystal structures have been determined for the processivity factor UL42 from the alphaherpesvirus herpes simplex virus type 1 (HSV-1) and for UL44 from the betaherpesvirus human cytomegalovirus (HCMV) (2, 3, 58). Despite having little if any sequence homology with processivity factors outside of their herpesvirus subfamily, these structures all share the “processivity fold” originally seen in the structure of the bacterial beta clamp (26). Interestingly, some of these processivity factors have a different quaternary structure. PCNA forms a head-to-tail trimeric ring (18, 27), HSV-1 UL42 is a monomer (10, 14, 16, 46, 58) equivalent to one-third of the PCNA complex, and HCMV UL44 is a head-to-head dimer in the form of a C-shaped clamp (2, 3, 9).Both HSV-1 UL42 and HCMV UL44 have a basic face that has been shown to be important for interacting with DNA (25, 35). In the case of dimeric HCMV UL44, the basic surface of each monomer faces inward, toward the center of the C clamp, and includes a basic loop, called the “gap loop,” that is thought to wrap around DNA (24). Recently the crystal structure of the bacterial beta clamp was determined in complex with DNA (15). In that structure, DNA was found to be located in the central pore of the clamp. Amino acid residues that interacted with DNA were in positions structurally homologous to those found on the positively charged faces of UL42 and UL44.UL42 and UL44 each also has a surface, facing away from the DNA binding face, that is important for interacting with the catalytic subunit of the viral DNA polymerase. Indeed, both of these proteins have been crystallized in complex with C-terminal peptides from their respective catalytic subunits, HSV-1 UL30 and HCMV UL54 (2, 58). Together with biochemical and mutational analyses, these crystal structures indicated that, although the details of the interaction are different, the catalytic subunit of the polymerase binds to a region including and in close proximity to a long loop that connects the N- and C-terminal subdomains, called the interdomain connector loop (32-34). The corresponding region of PCNA is also important for polymerase attachment and mediates the interactions of PCNA with many other cellular proteins (40). Both UL54 and UL30 were shown to attach to their respective subunits, UL44 and UL42, by way of their extreme C termini. The C-terminal residues responsible for this interaction correspond to amino acids that are not detectably conserved, either by sequence or by structure, among herpesvirus catalytic subunits. The HSV-1 UL30-UL42 interaction involves a groove to one side of the UL42 connector loop, with hydrophilic interactions being critical (58). The HCMV UL54-UL44 interaction involves a crevice near the UL44 connector loop, and hydrophobic interactions are crucial (2, 32, 33). Moreover, the HCMV UL44 crevice is on the opposite side of the connector loop with respect to the HSV-1 UL42 groove.Kaposi''s sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus, encodes a viral DNA polymerase catalytic subunit, Pol-8, and an accessory subunit, PF-8 (4, 7, 8, 29, 48, 57). PF-8 can bind to Pol-8 directly and specifically (8, 29) and is required for long-chain DNA synthesis in vitro (29). Similarly to UL44, PF-8 forms dimers in solution and when bound to DNA (9). Although it is likely that UL44 and PF-8 are the processivity factors for HCMV and KSHV, respectively, rigorous experiments demonstrating this have not been performed. However, for the sake of brevity and clarity, we will refer to these proteins as processivity factors.Here we present the crystal structure of PF-8 and show that PF-8 forms a head-to-head homodimer akin to UL44 but lacking the long gap loops which are thought to wrap around DNA. This suggests that PF-8 binds DNA differently than does UL44 or UL42. Because Pol-8 appears to lack a long, flexible C-terminal tail with a length comparable to those of other herpesvirus Pols, we expect the mode of binding of the catalytic subunit to be different as well. Based on structural data, information from homologs, and initial biochemical results, we were able to identify possible sites for interactions with DNA and Pol-8 and to propose a model for the simultaneous interaction of all three components of the complex. Further, the availability of crystal structures for all three herpesvirus classes provides new insights into comparative structure, function, and evolution.     

A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.Human cytomegalovirus (HCMV) infects a broad spectrum of cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (6, 28, 36). Infection of this diverse spectrum of cell types contributes to the multiplicity of CMV-associated disease. HCMV infection of hepatocytes and epithelial cells in the gut and lungs following transplant immunosuppression is directly associated with CMV disease (3, 44). HCMV can be transported in the blood by monocyte-macrophages, and virus produced in these cells can infect endothelial cells, leading to virus spread into solid tissues such as the brain, liver, and lungs, etc. (16). Despite the broad spectrum of cells infected in vivo, propagation of HCMV in the laboratory is largely limited to normal human fibroblasts because other cells produce little virus. HCMV rapidly adapts to laboratory propagation in fibroblasts, losing the capacity to infect other cell types, i.e., epithelial and endothelial cells and monocyte-macrophages (9, 16, 18, 43). This adaptation to fibroblasts involves mutations in the unique long b′ (ULb′) region of the HCMV genome, which includes 22 genes (9). Targeted mutation of three of the ULb′ genes, UL128, UL130, and UL131, abolished HCMV infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (17, 18). Restoration of UL128-131 genes in HCMV laboratory strain AD169 (which cannot infect epithelial and endothelial cells) produced viruses capable of infecting these cells (18, 48). There is also evidence that the UL128-131 proteins are deleterious to HCMV replication in fibroblasts, resulting in rapid loss or mutation of one or more of the UL128-131 genes during passage in fibroblasts (2).A major step forward in understanding how the UL128-131 genes promote HCMV infection of epithelial and endothelial cells involved observations that the UL128-131 proteins assemble onto the extracellular domain of the membrane-anchored HCMV glycoprotein heterodimer gH/gL (1, 49). Antibodies to UL128, UL130, and UL131 each neutralized HCMV for infection of endothelial or epithelial cells (1, 49). All herpesviruses express gH/gL homologues and, where this has been tested, all depend upon gH/gL for replication and, more specifically, for entry into cells (14, 15, 31, 38). Indeed, we showed that the gH/gL/UL128-131 complex mediated entry into epithelial and endothelial cells (40). All five members of the gH/gL/UL128-131 complex were required for proper assembly and export from the endoplasmic reticulum (ER) and for function (39, 41). In addition, the expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV entry into these cells (39). This interference suggested that there are saturable gH/gL/UL128-131 receptors present on epithelial cells, molecules that HCMV uses for entry. There was no interference in fibroblasts expressing gH/gL/UL128-131, although some interference was observed with gH/gL (39). As noted above, gH/gL/UL128-131 plays no obvious role in entry into fibroblasts and, in fact, appears to be deleterious in this respect (2, 18, 40).HCMV also expresses a second gH/gL complex, as follows: gH/gL/gO (20, 21, 22, 30, 48). Fibroblast-adapted HCMV strain AD169 expresses a gO protein that is a 110- to 125-kDa glycoprotein (21). Pulse-chase studies suggest that gH/gL assembles first in the ER before binding and forming disulfide links with gO (21, 22). The 220-kDa immature gH/gL/gO complex is transported from the ER to the Golgi apparatus and increases in size to ∼280 to 300 kDa before incorporation into the virion envelope (21). gH/gL/gO complexes are apparently distinct from gH/gL/UL128-131 complexes because gO-specific antibodies do not detect complexes containing either UL128 or UL130 and UL128-specific antibodies do not precipitate gO (49). Towne and AD169 gO-null mutant laboratory strains can produce small plaques on fibroblasts, leading to the conclusion that gO is not essential. However, the AD169 and Towne mutants produced ∼1,000-fold less infectious virus than wild-type HCMV (14, 19), which might also be interpreted to mean that gO is very important or even essential for replication. Thus, the prevailing model has been that wild-type HCMV particles contain the following two gH/gL complexes: gH/gL/gO, which promotes infection of fibroblasts, and gH/gL/UL128-131, which promotes entry into epithelial and endothelial cells. Supporting this model, there are two different entry mechanisms, as follows: HCMV enters fibroblasts by fusion at the plasma membrane at neutral pH (12), whereas entry into epithelial and endothelial cells involves endocytosis and a low pH-dependent fusion with endosomes (40). This model of HCMV entry parallels models for Epstein-Barr virus (EBV) entry that use gH/gL to enter epithelial cells and gH/gL/gp42 to enter B cells (24). Similarly, HHV-6 uses gH/gL/gO and gH/gL/gQ, which bind to different receptors (33).Many of the studies of gH/gL/gO have involved the fibroblast-adapted HCMV strain AD169, which fails to express UL131 and assemble gH/gL/UL128-131 or AD169 recombinants in which UL131 expression was restored (20, 21, 22, 48, 49). It seemed possible that the adaptation of AD169 to long-term passage in fibroblasts might also involve alterations in gO. HCMV gO is unusually variable (15 to 25% amino acid differences) among different HCMV strains compared with other viral genes (13, 34, 35, 37, 46). In recent studies, Jiang et al. (26) described a gO-null mutant derived from the HCMV strain TB40/E, a strain that can infect endothelial cells following extensive passage on these cells. The TB40/E gO-null mutant spread poorly on fibroblasts compared with wild-type TB40/E, and there was little infectious virus detected in fibroblast culture supernatants. However, the few TB40/E gO-null mutant particles produced by fibroblasts that could initiate infection of endothelial cells were able to spread to form normal-sized plaques on endothelial cells. These results further supported the model for which gH/gL/gO is required for infection of fibroblasts but not for epithelial/endothelial cells. Those authors also concluded that gO is important for the assembly of enveloped particles in fibroblasts, based on observations of few infectious virus particles in supernatants and cytoplasmic accumulation of unenveloped capsids (26).Our studies of gH/gL/UL128-131 have involved the clinical HCMV strain TR (39, 40, 41, 47). HCMV TR was originally an ocular isolate from an AIDS patient (45) and was passaged only a few times on fibroblasts before being genetically frozen in the form of a bacterial artificial chromosome (BAC) (34, 40). HCMV TR infects epithelial and endothelial cells (40) and monocyte-macrophages (D. Streblow and J. Nelson, unpublished results) well. In the accompanying paper (42), we characterized the biochemistry and intracellular trafficking of TR gO. TR gO expressed either in TR-infected cells or by using adenovirus vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of gO with gH/gL promoted transport of gH/gL beyond the ER. Importantly, TR gO was not found in extracellular virions. In contrast, AD169 gO was present in extracellular virus particles, as described previously (20, 21). We concluded that TR gO is a chaperone that promotes ER export of the gH/gL complex, but gO dissociates prior to incorporation into the virus envelope. Moreover, these differences highlight major differences between gO molecules expressed by fibroblast-adapted strain AD169 and low-passage TR.To extend these results and characterize how TR gO functions, whether in virus entry or virus assembly/egress, we constructed a TR gO-null mutant. TRΔgO exhibited major defects in entering fibroblasts, as evidenced by increased virus infection following treatment with the chemical fusogen polyethylene glycol (PEG). Unexpectedly, the mutant also failed to enter epithelial and endothelial cells, and again, PEG partially restored entry. Relatively normal numbers of TRΔgO particles were produced and released into cell culture supernatants, although even with PEG treatment, most of these virus particles remained defective in initiating immediate-early HCMV protein synthesis. Western blot analyses of TRΔgO extracellular particles demonstrated very low levels of gH/gL incorporated into virions, which likely explains the reduced entry of TRΔgO. However, the small amounts of gH/gL complexes that were present in TRΔgO virions were associated with increased quantities of UL130, and these TRΔgO particles spread better than wild-type HCMV on epithelial cell monolayers. Together with the results shown in the accompanying paper (42), we concluded that HCMV TR gO functions as a chaperone to promote ER export of gH/gL to HCMV assembly compartments and the incorporation of gH/gL into the virion envelope. The highly reduced quantities of gH/gL in virions are apparently responsible for the inability of HCMV to enter fibroblasts and epithelial and endothelial cells. These results suggest a modified version of our model, in which gH/gL, not gH/gL/gO, mediates entry into fibroblasts and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

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).     

Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1

Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G₁/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.Human cytomegalovirus (HCMV), a highly prevalent β-herpesvirus, can cause serious birth defects and disease in immunocompromised individuals, and it may be associated with cancer and cardiovascular disease (53). Viral gene expression is temporally regulated and is dependent on many cellular factors for a productive infection. Immediate early (IE) genes are expressed by 2 h postinfection (p.i.) and transactivate the early genes required for viral DNA replication. The expression of the late genes, which encode proteins involved in virion maturation and egress, is dependent on viral DNA replication.The virus has adopted different strategies for altering the cellular environment to make it more conducive to productive infection, including the stimulation of host cell DNA replication pathways, cell cycle deregulation and arrest, immune evasion, and inhibition of apoptosis (53). Although HCMV encodes its own DNA polymerase, it is dependent on other cellular resources for DNA replication. Infection of quiescent cells induces passage toward S phase such that the host cell is stimulated to generate proteins and DNA precursors necessary for genome replication; however, entry into S phase and cellular DNA replication are subsequently blocked and the cell arrests in G₁/S (1, 10, 11, 14, 30, 45). Cellular resources are thereby presumably free to be efficiently utilized for viral replication. Cell cycle arrest by HCMV is achieved in part through the misregulation of several cell cycle proteins, including the phosphorylation and accumulation of the Rb family pocket proteins, upregulation of cyclins E and B and their associated kinase activities, inhibition of cyclin A expression, stabilization of p53, and accumulation of Cdc6 and geminin, which inhibits licensing of the cellular origins of DNA replication (8, 17, 30, 49, 54, 65). Some of these cell cycle defects can be attributed to a deregulation of the anaphase-promoting complex (APC) (8, 72, 79, 80), an E3 ubiquitin ligase that is responsible for the timely degradation of cell cycle proteins and mitotic cyclins to promote cycle progression from mitosis through G₁ to S phase (58, 74). As the APC also appears to be a common target among other viruses, including the chicken anemia virus, adenoviruses, and poxviruses (23, 36, 52, 70), understanding the mechanisms leading to its inactivation during viral infection has been of great interest.As we have previously reported, multiple mechanisms may be involved in disabling the APC during HCMV infection (72), which is not surprising given the complexity of its structure and regulation (for a review, see references 58 and 74). The APC is a large multisubunit complex consisting of at least 11 conserved core subunits, as well as other species-specific subunits. In metazoans, the APC2 and APC11 subunits form the catalytic core, and along with APC10, provide the platform for binding the E2 ubiquitin-conjugating enzyme. Each of the APC3, APC8, APC6, and APC7 subunits contain multiple copies of the tetratricopeptide repeat (TPR) motif and together make up the TPR subcomplex, which provides a platform of protein interaction surfaces for binding the coactivators (i.e., Cdh1 and Cdc20) and various substrates. These two subcomplexes are bridged by the large scaffolding subunit APC1, with the TPR subcomplex tethered to APC1 through APC4 and APC5. The binding between APC1, APC4, APC5, and APC8 is also interdependent, such that the loss of one subunit decreases the association of the other three (71).The APC is activated by either of its coactivators, Cdh1 or Cdc20, which also function in recruiting specific substrates to the APC during different phases of the cell cycle. The phosphorylation of several APC subunits at the onset of mitosis, including APC1 and the TPR subunits, by cyclin B/cyclin-dependent kinase 1 (Cdk1) and Plk1 allows the binding of Cdc20 and subsequent activation of the APC (APC^Cdc20) (19, 37), whereas the binding and activation of the complex by Cdh1 is inhibited through its phosphorylation by cyclin B/Cdk1 (9, 29, 38, 83). As cells pass the spindle assembly checkpoint, APC^Cdc20 ubiquitinates securin (to allow for sister chromatid separation) and cyclin B for degradation by the proteasome (42, 67). The subsequent inactivation of Cdk1 and activation of mitotic phosphatases during late anaphase relieves the inhibitory phosphorylation on Cdh1, presumably by Cdc14 (6, 38, 44), which then allows Cdh1 to bind and activate the APC (APC^Cdh1). APC^Cdh1 ubiquitinates Cdc20 and mitotic cyclins for degradation to facilitate mitotic exit and maintains their low levels, along with S-phase regulators (e.g., Cdc6, geminin, etc.), during G₁ (16, 50, 59, 63). The inactivation of APC^Cdh1 as cells enter S phase may be mediated in part through the phosphorylation of Cdh1 by cyclin A/Cdk2 (46) and Cdh1 binding to the inhibitor Emi1 (25). The inactivation of Cdh1 by phosphorylation has been shown in all organisms studied thus far (e.g., yeast, Drosophila, plants, mammals, etc.), and mutants mimicking constitutively phosphorylated Cdh1 on Cdk consensus sites can neither bind nor activate the APC in vivo or in vitro (9, 29, 38, 69, 83).During HCMV infection of fibroblasts in G₀/G₁, however, Cdh1 becomes prematurely phosphorylated in a Cdk-independent manner and no longer associates with the APC (72). This dissociation does not appear to be due to an overexpression of Emi1 (79). Cdc20 also can no longer associate with the APC (79), suggesting a defect in the APC core. We have further shown that the APC core complex disassembles during the infection, with the TPR subunits (i.e., APC3, APC7, and APC8) and APC10 localizing to the cytosol, while APC1 remains nuclear (72). Interestingly, both the phosphorylation of Cdh1 and the dissociation of the APC occur at similar times during HCMV infection. Although either of these mechanisms could render the APC inactive, it was unclear whether these processes are linked or represent independent (or redundant) pathways. The causative factor(s) in mediating these events and the question of whether such a factor(s) was of cellular or viral origin also remained unresolved.On the basis of the results of several recent studies (26, 32, 62), the viral protein kinase UL97 emerged as a likely candidate for involvement in the phosphorylation of Cdh1. Conserved among herpesviruses, UL97 functions in viral genome replication (7, 32, 81) and in nuclear egress of viral capsids (21, 39, 48). UL97 is present in the tegument of the virus particle (76) and is also expressed de novo with early kinetics (i.e., detectable by 5 h p.i. by Western blot assay), with increased expression at later times of the infection (51, 76, 77). UL97 is a serine/threonine (S/T) protein kinase (22), and recent studies have further characterized it as a Cdkl mimic, with predicted structural similarity to Cdk2 (64) and common substrates. UL97 has been shown to phosphorylate in vitro nuclear lamin A/C (21), the carboxyl-terminal domain of RNA polymerase II (5), the translation elongation factor 1δ (EF1δ) (33), and Rb (26, 62) on sites targeted by Cdks, and there is considerable evidence that UL97 phosphorylates lamin A/C, EF1δ, and Rb on these sites in infected cells as well (21, 26, 33, 62). Given that cyclin A/Cdk2 and cyclin B/Cdk1 complexes normally phosphorylate Cdh1, thus preventing its association with the APC, we hypothesized that UL97 phosphorylates Cdh1 during HCMV infection.In the present study, we provide further mechanistic details of the events and players involved in inactivating the APC during HCMV infection. Evidence that UL97 is the viral factor mediating the phosphorylation of Cdh1 was obtained. However, APC disassembly still occurred at similar times in ΔUL97 and wild-type virus infections, indicating that UL97-mediated phosphorylation of Cdh1 is not required for this event. The inactivation of the APC core complex is further attributed to the loss of the APC5 and APC4 subunits early during the infection. The degradation of these subunits is proteasome dependent and requires de novo synthesis of viral early or cellular proteins. While the primary mechanism of inactivation appears to be the dissociation of the complex and the targeted loss of APC5 and APC4, phosphorylation of Cdh1 may provide a small kinetic advantage and backup mechanism for disabling the APC.     

Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids

Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229→L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.Egress of herpesvirus capsids from the nucleus occurs by envelopment of capsids at the inner nuclear membrane (INM) and is followed by de-envelopment at the outer nuclear membrane (ONM). This process can be broken down into a pathway of discrete steps that begin with recruitment of the viral envelopment apparatus to the INM. Herpes simplex virus type 1 (HSV-1) UL34 and UL31 and their homologs in other herpesviruses are required for efficient envelopment at the INM (7, 13, 22, 23, 29). HSV-1 pUL31 and pUL34 are targeted specifically to the INM by a mechanism that requires their interaction with each other (27, 28), and this mutual dependence is a conserved feature of herpesvirus envelopment (9, 14, 27, 28, 32, 33, 39). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane (22, 24, 30). The sequences in HSV-1 pUL34 that mediate interaction with UL31 and that lead to nuclear envelope targeting were mapped to amino acids (aa) 137 to 181 (16). The sequences in the murine cytomegalovirus (MCMV) homolog of UL31, M53, that mediate the nuclear envelope targeting interaction with the UL34 homolog, M50, were mapped to the N-terminal third of the protein in the first of four conserved regions (17), and Schnee et al. subsequently showed that this same region of pUL31 homologs from other families of herpesviruses mediates interaction with the corresponding pUL34 homologs (33).After the targeting of the pUL34/pUL31 complex to the INM, subsequent steps in nuclear egress include, it is thought, (i) local disruption of the nuclear lamina to allow capsid access to the INM, (ii) recognition and docking of capsids by the envelopment apparatus at the INM, (iii) curvature of the inner and outer nuclear membranes around the capsid, (iv) scission of the INM to create an enveloped virion in the space between the INM and ONM, (v) fusion of the virion envelope with the outer nuclear membrane, and (vi) capsid release into the cytoplasm.At least some of the viral and cellular factors critical for nuclear lamina disruption and for de-envelopment fusion have been identified. pUL34, pUL31, and pUS3 of HSV-1 have all been implicated in changes in localization, interaction, and phosphorylation of nuclear lamina components, including lamins A/C and B and the lamina-associated protein, emerin (3, 15, 19, 20, 24, 26, 34, 35). pUS3, pUL31, and glycoproteins B and H have been implicated in de-envelopment of primary virions at the ONM (8, 21, 28, 30, 38).pUL34 and pUL31 are thought to be involved in steps between lamina disruption and de-envelopment, but genetic evidence in infected cells has so far been lacking. Klupp et al. have shown that overexpression of alphaherpesvirus pUL31 and pUL34 in the absence of other viral proteins can induce formation of small vesicles derived from the INM, suggesting a role for these two proteins in membrane curvature around the capsid (12). Tight membrane curvature is an energetically unfavorable event and is thought to be accomplished by coupling curvature to energetically favorable interactions between membrane-bound proteins or protein complexes (reviewed in reference 40). The data of Klupp et al. suggest the possibility that upon recognition of a capsid, pUL31 and pUL34 may interact in a way that induces tight curvature of the INM. Here we present data in support of this hypothesis, showing that a specific point mutation in UL34 induces accumulation of docked capsids at the INM, extragenic suppression of the mutant phenotype is associated with a mutation in UL31, and pUL31 and pUL34 can interact via sequences that are not involved in their INM targeting interaction.We previously published a characterization of a library of 19 charge cluster mutants of pUL34. In each of these mutants, one charge cluster (defined as a group of five consecutive amino acids in which two or more of the residues have charged side chains) was mutated such that the charged residues were replaced by alanine. Six of the 19 charge cluster mutants tested failed to complement replication of UL34-null virus, indicating that they disrupt essential functions of pUL34. Interestingly, five of the six noncomplementing mutants were synthesized at levels comparable to that of wild-type UL34 and localized normally to the nuclear envelope, suggesting that they were unimpaired in their ability to make a nuclear envelope targeting interaction with UL31. In order to identify essential functions of pUL34 downstream of nuclear envelope targeting, we have undertaken a detailed study of the behavior and interactions of these mutants.     

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.     

Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.Human cytomegalovirus (HCMV) infects many different cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, smooth muscle cells, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (reviewed in references 5, 30, 38, and 45). In the laboratory, HCMV is normally propagated in primary human fibroblasts because most other cell types yield low titers of virus. Commonly studied laboratory strains, such as AD169, were propagated extensively in fibroblasts, and this was accompanied by deletions or mutations in a cluster of 22 genes known as ULb′ (6). These mutations were correlated with the inability to infect other cell types, including endothelial and epithelial cells and monocyte-macrophages. Targeted mutagenesis of three of the ULb′ genes, UL128, UL130, and UL131, abolished infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (13, 15). Restoration of the UL128-131 genes in laboratory strains of HCMV strains restored the capacity to infect endothelial and epithelial cells and other cells (15, 52).The UL128, UL130, and UL131 proteins assemble onto the extracellular domain of HCMV gH/gL (1, 42, 53). For all herpesviruses, gH/gL complexes mediate entry into cells (12, 33, 39), suggesting that gH/gL/UL128-131 might participate in the entry mechanism. Indeed, we demonstrated that gH/gL/UL128-131 mediates entry into epithelial and endothelial cells by using the fusogenic agent polyethylene glycol to force entry of HCMV UL128-131 mutants into these cell types (41). This was consistent with reports that UL128-, UL130-, and UL131-specific antibodies blocked the capacity of HCMV to infect epithelial and endothelial cells but not fibroblasts (1, 53). Furthermore, expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV infection, consistent with saturable gH/gL/UL128-131 receptors (40). Expression of all five proteins was necessary so that the gH/gL/UL128-131 complexes were exported from the endoplasmic reticulum (ER) and could function (40-42, 53). Together, these data suggested that gH/gL/UL128-131 mediates entry into epithelial/endothelial cells but is not required for entry into fibroblasts. By extension, it was reasonable to propose that other forms of gH/gL might facilitate the entry into fibroblasts.The laboratory HCMV strain AD169 is known to express a second gH/gL complex containing glycoprotein O (gO) (21-23, 53). In cells infected with a recombinant AD169 in which the UL131 mutation was repaired, gH/gL/gO complexes were separate from gH/gL/UL128-131 complexes, i.e., gO was not detected following immunoprecipitation (IP) with UL128- and UL130-specifc antibodies, and gO-specific antibodies did not precipitate UL128 and UL130 (53). AD169 and Towne gO⁻ mutants produce small plaques on fibroblast monolayers and low titers of virus, supporting an important, although not essential, role for gH/gL/gO in virus replication in fibroblasts (11, 19). AD169 does not infect endothelial and epithelial cells, so AD169 gO⁻ mutants were not tested on these cells. Jiang et al. described a gO-null mutant derived from an endotheliotropic HCMV strain, TB40/E (27). The TB40/E gO-null mutant spread normally on endothelial cells, suggesting that gO or gH/gL/gO is less important for infection and spread in these cells. Given that the role of gH/gL in entry is highly conserved among the herpesviruses, it seemed likely that gH/gL/gO might be involved in entry into fibroblasts. Consistent with this notion, Paterson et al. showed that anti-gO antibodies decreased fusion from without caused by infection of cells with HCMV AD169 (37). These observations supported our working model in which gH/gL/UL128-131 mediates entry into epithelial and endothelial cells, while gH/gL/gO mediates entry into fibroblasts. There is also the possibility that gH/gL (lacking gO and UL128-131) might be incorporated into the virion envelope, although there is presently no direct evidence for this. Any gH/gL detected biochemically might result from dissociation of gO or UL128-131 during sample preparation and analysis. gH/gL expressed without other HCMV proteins was retained in the ER (42), arguing against incorporation into the virion.Other herpesviruses, e.g., Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-7, use different forms of gH/gL to enter different cell types via different pathways (25, 34, 43). Similarly, HCMV entry into fibroblasts occurs by fusion at the plasma membrane at a neutral pH and does not require gH/gL/UL128-131 (7), whereas entry into epithelial and endothelial cells involves endocytosis and low pH-dependent fusion and requires gH/gL/UL128-131 (41).All of the biochemical analyses of gO in terms of binding to gH/gL and intracellular transport have involved fibroblast-adapted strain AD169 (21-23, 31, 53). These studies indicated that gO is a 110- to 125-kDa glycoprotein encoded by the UL74 gene (22). Glycosidase digestion experiments demonstrated that the gO polypeptide chain is ∼62 to 65 kDa (21-23, 53). Pulse-chase studies showed that gH/gL assembles in the ER as a disulfide-linked heterodimer (28) that subsequently binds to, and establishes disulfides with, gO (22, 23). The 220-kDa immature gH/gL/gO trimer is initially sensitive to endoglycosidase H (endo H), which removes immature N-linked oligosaccharides from glycoproteins present in the ER (22, 23). Transport of gH/gL/gO to the Golgi apparatus is associated with processing of N-linked oligosaccharides to mature forms that resist endo H. Also associated with transport to the Golgi apparatus is the addition of O-linked oligosaccharides and phosphorylation, increasing the molecular weight of gO (after reduction) to 125 to 130 kDa and that of the gH/gL/gO complex to 240 to 260 kDa (22, 23, 29). It is the mature glycoprotein complex, previously known as gCIII, that is trafficked to HCMV assembly compartments for incorporation into the virion envelope (22, 23, 29).In addressing the function of gO, it is important to recognize that AD169 has adapted to replication in fibroblasts, losing expression of UL131 and failing to assemble gH/gL/UL128-131 complexes (6) (15). There seems to be strong pressure to mutate UL128-131, because clinical strain Merlin acquired a UL128 mutation within 5 passages on fibroblasts (2). It is also reasonable to suggest that fibroblast adaptation includes changes in gO. The gO genes (UL74) of several laboratory and clinical strains and clinical isolates are highly variable (up to 25% of amino acids) (10, 35, 37, 47). However, it is important to note that AD169-derived UL131-repair virus can infect epithelial and endothelial cells (52). Thus, if AD169 gO is important for infection of these cells, then gO must be functionally normal in this regard. These differences in laboratory versus clinical HCMV prompted us to characterize the gO molecule expressed by the HCMV strain TR. HCMV TR is a clinical isolate that was stabilized in the form of a bacterial artificial chromosome (BAC) after very limited passage in fibroblasts (35, 41). HCMV TR expresses gH/gL/UL128-131 (42) and infects epithelial and endothelial cells (41) and monocyte-macrophages well (D. Streblow and J. Nelson, unpublished results).Here, we report our biochemical and cell trafficking analyses of the TR gO protein. We were surprised to find that TR gO was not present in extracellular virus particles. In contrast, gO was detected in extracellular AD169 particles, consistent with previous findings (22). TR gO expressed either in HCMV-infected cells or by using nonreplicating Ad vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of TR gO with gH/gL promoted transport of gH/gL beyond the ER, and gO was slowly lost from gH/gL complexes but not secreted from cells and not observed in extracellular virus particles. Thus, TR gO acts as a chaperone. Consistent with this, in the accompanying paper by Wille et al. (54), a TR gO-null mutant was described that secreted extracellular particles containing markedly reduced quantities of gH and gL. The gO⁻ mutant failed to enter fibroblasts and also epithelial and endothelial cells. Together, these results suggest that it is gH/gL, not gH/gL/gO, which is incorporated into HCMV TR virions. It appears that gH/gL is required for entry into fibroblasts, and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

The interactions of the herpes simplex virus processivity factor UL42 with the catalytic subunit of the viral polymerase (Pol) and DNA are critical for viral DNA replication. Previous studies, including one showing that substitution of glutamine residue 282 with arginine (Q282R) results in an increase of DNA binding in vitro, have indicated that the positively charged back surface of UL42 interacts with DNA. To investigate the biological consequences of increased DNA binding by UL42 mutations, we constructed two additional UL42 mutants, including one with a double substitution of alanine for aspartic acid residues (D270A/D271A) and a triple mutant with the D270A/D271A and Q282R substitutions. These UL42 mutants exhibited increased and prolonged DNA binding without an effect on binding to a peptide corresponding to the C terminus of Pol. Plasmids expressing any of the three UL42 mutants with an increased positive charge on the back surface of UL42 were qualitatively competent for complementation of growth and DNA replication of a UL42 null mutant on Vero cells. We then engineered viruses expressing these mutant proteins. The UL42 mutants were more resistant to detergent extraction than wild-type UL42, suggesting that they are more tightly associated with DNA in infected cells. All three UL42 mutants formed smaller plaques on Vero cells and replicated to reduced yields compared with results for a control virus expressing wild-type UL42. Moreover, mutants with double and triple mutations, which contain D270A/D271A mutations, exhibited increased mutation frequencies, and mutants containing the Q282R mutation exhibited elevated ratios of virion DNA copies per PFU. These results suggest that herpes simplex virus has evolved so that UL42 neither binds DNA too tightly nor too weakly to optimize virus production and replication fidelity.Processivity factors of DNA polymerases promote long-chain DNA synthesis by preventing dissociation of the DNA polymerase from the primer/template. Processivity factors also can influence DNA replication fidelity, as indicated by numerous in vivo and in vitro studies (1-3, 5, 6, 11, 12, 18, 28, 36). A major class of processivity factors known as “sliding clamps” includes proliferating cell nuclear antigen (PCNA) of eukaryotic cells (23) and gp45 of T4 bacteriophage (27). Sliding clamps are homodimers or homotrimers that encircle DNA and interact with the catalytic subunits (Pols) of their cognate DNA polymerases to promote processive DNA synthesis.A second class of processivity factors includes those encoded by herpesviruses and is exemplified by herpes simplex virus (HSV) UL42. UL42 forms a heterodimer with the HSV Pol. Both subunits are essential for production of infectious virus and for viral DNA replication (20, 26). UL42 can stimulate long-chain DNA synthesis by Pol, and template challenge experiments established that this stimulation is due to increased processivity (15). In addition to its interaction with Pol, which is mediated by the C terminus of Pol, UL42 also binds DNA directly with high affinity (14, 15, 30, 37). This mode of DNA binding differs from that of sliding clamps, which do not form high-affinity direct interactions with DNA (13) but must be loaded onto DNA with the aid of ATP-dependent clamp loaders for their normal functioning (16). Nevertheless, the structure of UL42 is very similar to a monomer of the sliding clamp PCNA (39). Like other processivity factors, UL42 also plays a role in maintaining DNA replication fidelity both in vivo and in vitro (5, 18).The “back face” (opposite face to the side that binds Pol) of a UL42 molecule contains several positively charged residues. By titrating the effects of cations on UL42 DNA binding, it was determined that charge-charge interactions are involved in the interaction (22). Substitutions of alanine for any of four arginine residues on the back face of UL42 resulted in substantial reductions in DNA binding without affecting the binding to peptide corresponding to the C terminus of Pol in vitro (31), while substitutions of lysine for arginine had little or no effect on DNA binding affinity (22). A UL42 mutant (Q282R) containing a substitution of arginine for a negatively charged glutamine residue on the back face of UL42 exhibited a fourfold increase in DNA binding without altering the interaction with the Pol C-terminal peptide in vitro (22). Therefore, the positively charged surface of UL42 is important for the interaction between UL42 and DNA. A question raised by these studies is whether UL42 could bind DNA so tightly as to affect HSV replication.Mutant viruses engineered to encode individual arginine-to-alanine substitution mutations in UL42 exhibit several phenotypes, including a delayed onset of viral DNA replication, reduced virus yields, and reduced fidelity of DNA replication (18). Recombinant viruses expressing UL42 with multiple substitutions of alanine for arginine residues exhibit even greater effects on viral DNA replication and virus yields (19). Thus, reducing DNA binding by UL42 deleteriously affects viral growth and DNA replication fidelity. However, these studies did not address whether increasing DNA binding by UL42 would have any effects on viral DNA replication, replication fidelity, or virus production.In this study we engineered two new UL42 mutant proteins (with the D270A/D271A or Q282R/D270A/D271A mutations) that contain less negative charge on the back face and examined the effects of these substitutions on DNA and Pol peptide binding. In addition, recombinant viruses were constructed to examine the effect of these multiple substitutions and the single Q282R substitution on virus production, DNA replication, and the fidelity of DNA replication.