Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.Human cytomegalovirus (HCMV) is a widespread betaherpesvirus that causes lifelong persistent infections with occasional reactivations. While HCMV infection is usually clinically unapparent in the immunocompetent host, it can cause severe disseminated infections under conditions of immunosuppression, with manifestations in the lung, retina, and gastrointestinal tract, among others (12). Various cell types support viral replication, including epithelial cells and endothelial cells (ECs), smooth muscle cells, fibroblasts, and cells of hematopoietic origin (13, 14, 18, 19, 25, 26, 37). Among these target cells, endothelial cells are assumed to contribute particularly to hematogenous dissemination of HCMV (24).While recent clinical HCMV isolates are characterized by this broad cell tropism, the target cell range becomes restricted during long-term propagation on fibroblasts (28, 33). The underlying mechanism for this cell culture adaptation is a modulation within the viral genes UL128, UL130, and UL131A (8, 11). These three genes have been shown to be essential for infection of granulocytes, dendritic cells, epithelial cells, and endothelial cells but are dispensable for infection of fibroblasts (1, 9, 11, 34, 35). The encoded proteins pUL128, pUL130, and pUL131A were reported to form a complex with the viral glycoproteins gH and gL that is distinct from the glycoprotein complex gCIII (gH/gL/gO) (35). Whereas poorly endotheliotropic HCMV strains bear just the gH/gL/gO complex in their envelopes, highly endotheliotropic strains bear both gCIII variants: gH/gL/gO and gH/gL/pUL128-131A. Deletion of any of the three genes UL128-131A results in loss of EC tropism (11), most likely because only a complete complex of gH/gL and pUL128, pUL130, and pUL131A can efficiently function in endocytic entry in ECs (21). However, functional sites within the proteins (e.g., mediating binding to the viral complex partners or interaction with a putative cellular receptor) have not yet been identified. One approach to search for candidate protein-protein interaction sites is charge-cluster-to-alanine (CCTA) mutagenesis. This method is based on the assumption that clusters of charged amino acids tend to be exposed in the tertiary structure of a protein and are thus likely to be sites of interaction with other proteins. Replacement of these charged amino acids by uncharged alanines should then target protein-protein interaction sites without destroying the protein backbone (5, 7). Applying this method to HCMV pUL128, we were able to identify a central core region within pUL128 essential for EC infection as well as contributing sites in the N-terminal half and the C terminus of the protein (22). We now aimed to extend the study to the scanning of UL130 by markerless mutagenesis in the context of a highly endotheliotropic HCMV BACmid, TB40-BAC4. The resulting mutant viruses were then characterized with regard to their ability to infect ECs to identify the relevant parts of the protein.With regard to the role of UL130 in EC infection by endocytosis, the C-terminal part of pUL130 was of special interest. A frameshift mutation that changes the last 11 amino acids (aa) of pUL130 is the most prominent difference between the poorly endotheliotropic HCMV strain Towne and the highly endotheliotropic strain HCMV-TB40-BAC4 in this region (8, 11, 27). Rhee and Davis have described a cell-penetrating pentapeptide (CPP) motif (PFVYLI) mediating internalization by endocytosis, which is clathrin and caveolin independent but may involve lipid rafts (17). Not only do the last five amino acids of pUL130 (PNLIV) bear a striking similarity to this motif, but also the entry of HCMV into ECs has been reported to occur by an endocytic pathway (20, 23). Thus, we hypothesized that the pentapeptide motif PNLIV in pUL130 might be involved in mediating endocytic uptake of HCMV in ECs, and if so, deletion of this motif should result in a nonendotheliotropic virus. A number of CPPs that are thought to be taken up by endocytosis have now been described, including VPMLK, PMLKE, VPTLK, KLPVM, and others (32). These CPPs all bear some similarity, but the exact amino acid sequence seems to be irrelevant. We thus hypothesized for UL130 that a scrambled mutant (PNLIV changed to PINVL) should still be able to mediate endocytosis of HCMV in ECs. To test these assumptions we generated a series of mutant viruses where the PNLIV motif was either deleted, scrambled (PNLIV changed to PINVL), or exchanged against a known CPP (PFVYLI [17]) and characterized them with regard to EC infectivity.     

Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

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

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).     

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

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

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.     

Major Tegument Protein pp65 of Human Cytomegalovirus Is Required for the Incorporation of pUL69 and pUL97 into the Virus Particle and for Viral Growth in Macrophages

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.Human cytomegalovirus (HCMV), a member of the Betaherpesvirinae subfamily, is a threatening pathogen for immunocompromised patients, such as transplant recipients, AIDS patients, and conatally infected infants (15). HCMV infection of individuals with a compromised immune system causes considerable morbidity and mortality after primary infection or reactivation from latency.Mature HCMV virions comprise four distinct structures: core, capsid, tegument, and envelope. The nucleocapsid consists of the core containing the approximately 240-kb linear double-stranded DNA genome, which is embedded in an icosahedral capsid. Between the envelope, a cellularly derived lipid membrane containing viral glycoproteins, and the nucleocapsid, a protein layer called tegument (26), is located. The tegument of HCMV is composed of at least 25 viral proteins. Tegument proteins have been proposed to act in several processes, such as immune evasion (reviewed in reference 30), release of viral DNA into the nucleus (6), and initiation and regulation of the viral replication cycle (3, 7, 16, 31, 41). However, for many of the tegument proteins, the morphogenetic or regulatory functions are unknown. An increasing number of host cell proteins, e.g., cytoskeletal proteins such as α- and β-actin, have also been detected in HCMV particles (4, 39). In addition to infectious virions, HCMV-infected cells generate two types of aberrant particles: noninfectious enveloped particles (NIEPs) and dense bodies (DBs) (18). The protein composition and morphology of NIEPs are nearly identical to those of mature virions; however, their lack of an electron-dense DNA-containing core allows discrimination of NIEPs from infectious virions by electron microscopy (18). DBs are fusion-competent enveloped particles lacking a nucleocapsid. They are composed primarily of the tegument protein pp65 (ppUL83) (4, 18, 39).For a long time, the herpesvirus tegument has been considered to be unstructured. Data mainly from alphaherpesviruses indicate that morphogenesis depends on an intricate network of tegument protein-protein interactions (reviewed in reference 23). Interestingly, for most tegument proteins of alphaherpesviruses relevant for primary tegumentation and envelopment, homologues have been found in HCMV, whereas there is much less homology between the proteins involved in secondary tegumentation and envelopment. Cryoelectron microscopic analyses of herpesvirus particles, including HCMV, provide evidence for an icosahedral symmetry and protein-protein complexes forming substructures, at least for the innermost part of the tegument (11).Remarkably, the most abundant tegument protein and major constituent of extracellular virions, pp65, is not essential for virus replication in fibroblasts in vitro. Deletion of pp65 in HCMV strain AD169 causes a complete loss of DB formation, while production of infectious virus in fibroblasts appears to be unaffected (34). Wild-type virus particle-associated pp65 is rapidly translocated to the nuclei of infected cells after penetration of the incoming virus (4, 33). Newly synthesized pp65 accumulates in both nucleus and cytoplasm at later stages of infection. In all, the precise function of pp65 during infection is not clear.During HCMV infection, pp65 is a major antigen for cellular immune responses. Besides its function as a structural component of the virus, pp65 seems to be involved in manipulation of the host''s immune system. Recent reports provide evidence that pp65 is involved in subverting the host immune response by mediating a decreased expression of major histocompatibility complex class II molecules (27). Microarray studies demonstrating an increase in the cellular antiviral cytokine response during infection with a pp65 deletion mutant suggested that pp65 is involved in downmodulation of beta interferon and of a number of chemokines (1, 8). However, most recent work demonstrates that not pp65 but the immediate-early 2 (IE2) gene product IE86 is responsible for the block of beta interferon induction during HCMV infection and that IE86 expression is delayed in the pp65 deletion mutant due to a decreased expression of pp71 (36). It has also been shown that pp65 can directly interact with NKp30, the natural killer (NK) cell-activating receptor, and that this interaction leads to a general inhibition of the killing ability of NK cells (2). Because of the requirement of cell-free pp65, the relevance of this interaction during HCMV infection in vivo is not entirely clear and needs to be investigated in more detail.Another feature of pp65 is the ability to interact with cellular as well as viral proteins. The interaction of pp65 with the cellular Polo-like kinase 1 (Plk1) results in an incorporation of Plk1 into virus particles. Plk1 is able to phosphorylate pp65 in vitro (14). Recently, it has been shown that pp65 interacts directly with the viral protein kinase pUL97 (20). pUL97 seems to be required for normal intranuclear distribution of pp65. Inhibition of the pUL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase domain results in accumulation of pp65 in characteristic inclusions in the nuclei of infected as well as transfected cells (28).To extend our knowledge about pp65 and its function, we investigated the composition of endotheliotropic HCMV particles in the absence of the most abundant tegument protein, pp65. We hypothesized that other viral or cellular proteins might compensate for the lack of pp65 in virus particles, as described for tegument mutants of pseudorabies virus (25). The results presented here, using a pp65 stop codon mutant of the endotheliotropic HCMV strain TB40/E, demonstrate that in contrast to our hypothesis, incorporation of at least three other HCMV tegument proteins, pUL25, pUL69, and pUL97, is severely impaired when pp65 is lacking. For pUL69, a direct interaction with pp65 could be shown in infected as well as transfected cells. These results show that pp65 interacts with other viral tegument proteins during infection, which in turn is important for the incorporation of these proteins into mature virus particles. Finally, for the first time, we could show a cell-specific biological relevance of pp65 for growth of HCMV in monocyte-derived macrophages (MDM).     

Random Transposon-Mediated Mutagenesis of the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.The herpesvirus particle is composed of four structural elements. The DNA genome-containing core is enclosed in an icosahedral capsid, which, in turn, is embedded in a proteinaceous layer termed the tegument and enveloped by a cell-derived membrane containing viral glycoproteins (35). The tegument of the Alphaherpesvirinae contains more than 15 different viral and several cellular proteins and can be structurally and functionally separated into at least two layers: a capsid-proximal “inner” part and an envelope-associated “outer” part (reviewed in references 34 and 35). The largest tegument proteins in all herpesviruses analyzed so far are homologs of herpes simplex virus type 1 (HSV-1) pUL36, which are essential for viral replication. pUL36, its interaction partner, pUL37, and the pUS3 kinase are part of the inner tegument and remain associated with nucleocapsids during their transport along microtubules to the nuclear pore (2, 3, 19, 31). In contrast, other tegument proteins like pUL46, pUL47, and pUL49 rapidly diffuse in the cytoplasm after fusion of the virion envelope with the plasma membrane. Proteolytic cleavage of HSV-1 pUL36 after docking of the nucleocapsid to the nuclear pore appears to be required for release of viral DNA into the nucleus (22). Besides these roles early in infection, pUL36 also functions during later stages of replication in virion maturation. After assembly in the nucleus, nucleocapsids are translocated to the cytoplasm by budding at the inner nuclear membrane and fusion with the outer nuclear membrane (34). Although functional nuclear localization motifs have been described for pseudorabies virus (PrV) and HSV-1 pUL36 (1, 37), in PrV-infected cells, pUL36 was never detected in the nucleus but was added to nascent virions early after nuclear egress (18, 27, 31, 37). It has been suggested that pUL36 interacts either directly (9, 32, 42, 44) or indirectly via capsid-associated pUL25 (10) with the capsid shell starting the tegumentation process in the cytosol.In PrV, pUL36 is the only tegument protein which has been shown to be truly essential. It consists of 3,084 amino acids (aa), resulting in a molecular mass of more than 300 kDa (27). Deletion of pUL36 in HSV-1 and PrV abolished viral replication. Ultrastructurally, similar phenotypes with nonenveloped nucleocapsids present in the cytoplasm and the lack of extracellular particles indicated a defect in virion maturation in the cytoplasm (13, 16). Several functional domains have been identified in pUL36. The interaction domain of pUL36 with pUL37 (5, 16, 20, 27, 36, 42) could be located in the N-terminal part of PrV and HSV-1 pUL36 (16, 36) (Fig. ​(Fig.1).1). Deletion of the pUL37 binding site in PrV pUL36 (PrV-UL36BSF) resulted in a similar phenotype to deletion of pUL37 with an impairment of secondary envelopment in the cytoplasm (16, 26). Unlike in PrV, pUL37 is essential for replication in HSV-1 (14, 30).Open in a separate windowFIG. 1.Schematic overview of PrV pUL36 and corresponding insertion mutants. (A) Diagram of the PrV genome with the unique long (U_L) and unique short (U_S) regions as well as repeat regions (internal repeat, IR; terminal repeat, TR). The positions of BamHI restriction sites are indicated, and restriction fragments are numbered according to their size. (B) Schematic diagram of the UL36 open reading frame with conserved regions. Pfam analysis (4; http://www.sanger.ac.uk/Software/Pfam/) delineated two highly conserved PfamA domains within pUL36 homologs of herpesviruses of all three herpesvirus subfamilies [box I, Herpes_teg_N PrV (p)UL36, aa 11 to 178] and of alphaherpesviruses [box II, Herpes_UL36 PrV (p)UL36, aa 1000 to 1251] as well as PfamB domains (hatched rectangles) (6) (C) Known essential and nonessential regions in PrV pUL36. Nonessential regions are shown in gray, with the positions of the amino acids deleted in the corresponding constructs (6, 8). Deletions tested by Lee et al. (28) are shown below, marked by arrows. The essential C terminus is shown in black. Besides the N-terminal deletion Δ6-225, none of the truncated proteins was functional. (D) Predicted or identified motifs in pUL36: USP (Cys26), active-site cysteine of the deubiquitinating activity (24); pUL37 interaction domain (16, 27); NLS, nuclear localization signal (37); leucine zipper (27); and late domain motifs PPKY and PSAP (6). (E) Locations of linker insertions in pUL36 are indicated by arrows and the position of the amino acid immediately preceding the insertion. Insertions shown by arrows pointing upwards yielded functional proteins, while arrows pointing downwards indicate nonfunctional mutants. Insertions resulting in proteins which were impaired but not fully deficient in complementation are underlined. For orientation, the BamHI site separating BamHI fragments 1 and 2 is indicated.A second functional domain in the N terminus of pUL36 comprises a ubiquitin-specific cysteine protease (USP) activity which could be identified in all three herpesvirus subfamilies (24, 40, 41). Interestingly, the USP activity is not essential for virus replication in cell culture (7, 21, 25, 43). However, it is relevant for oncogenicity of Marek′s disease virus (MDV) (21) and for virion maturation and neuroinvasion of PrV (7, 8, 29).Several other regions in PrV pUL36 were deleted without abolishing virus replication (6, 8, 28). While deletion of nearly 1/3 of the protein in the C-terminal part (aa 2087 to 2981) had only a slight effect, deletion of a region containing two leucine zipper motifs impaired virus replication and spread more strongly (8). The highly conserved C-terminal 62 amino acids, except for the extreme C-terminal 6 amino acids, are essential for virus replication (6, 28). Due to the size of the protein, a more detailed mutagenesis analysis has, however, not yet been undertaken.Therefore, the aim of our study was to construct random insertion mutants of PrV pUL36 using transposon-mediated insertion mutagenesis resulting in a 5-amino-acid linker insertion. Mutant proteins were analyzed functionally in transient transfection assays for complementation, and stable recombinants were isolated and further characterized.     

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP¹⁷⁴]), which has been proposed to be directly involved in ppUL44''s interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Δloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Δloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Δloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.The Betaherpesviridae subfamily member human cytomegalovirus (HCMV) is a major human pathogen, causing serious disease in newborns following congenital infection and in immunocompromised individuals (28, 42). Replication of its double-stranded DNA (dsDNA) genome occurs in the nuclei of infected cells via a rolling-circle process mediated by 11 virally encoded proteins (32, 33), including a viral DNA polymerase holoenzyme, comprising a catalytic subunit, pUL54, and a proposed processivity factor, ppUL44 (14). ppUL44 is readily detectable in virus-infected cells as a 52-kDa phosphoprotein of 433 amino acids with strong dsDNA-binding ability (30, 45). Defined as a “polymerase accessory protein” (PAP) whose function is highly conserved among herpesviruses, ppUL44 is an essential factor for viral replication in cultured cells and hence represents a potential therapeutic target to combat HCMV infection (39). It is a multifunctional protein capable of self-associating (5, 10), as well as interacting with a plethora of viral and host cell proteins, including the viral kinase pUL97 (29), the viral transactivating protein pUL84 (15), the viral uracil DNA glycosylase ppUL114 (37), and the host cell importin α/β (IMPα/β) heterodimer, which is responsible for its transport into the nucleus (4). The activities of ppUL44 as a processivity factor, including the ability to dimerize, as well as bind to, pUL54 and DNA, reside in the N-terminal portion (26, 45), whereas the C terminus is essential for phosphorylation-regulated, IMPα/β-dependent nuclear targeting of ppUL44 monomers and dimers (4-6). Once within the nucleus, ppUL44 is thought to tether the DNA polymerase holoenzyme to the DNA, thus increasing its processivity (14).Recent studies have identified specific residues responsible for ppUL44 interaction with pUL54, as well as for the interaction with IMPα/β and homodimerization (4, 10, 27, 41). The crystal structure of ppUL44''s N-terminal domain (Fig. ​(Fig.1A)1A) reveals striking similarity to that of other processivity factors, such as proliferating cell nuclear antigen (PCNA) and its herpes simplex virus type 1 (HSV-1) homologue UL42 (10, 46). Unlike the PCNA trimeric ring, however, both ppUL44 and UL42, which bind to dsDNA as dimers and monomers, respectively, have an open structure, which is believed to be the basis for their ability to bind to dsDNA in the absence of clamp loaders and ATP (9, 10, 46). Both ppUL44 and UL42 share a very basic “back” face, which appears to be directly involved in DNA binding via electrostatic interactions (19, 22, 23, 38, 46). One striking difference between ppUL44 and UL42 is the presence on the former of an extremely basic flexible loop (UL44-FL, PHTRVKRNVKKAP¹⁷⁴) protruding from the basic back face of the protein (Fig. ​(Fig.1A).1A). Comparison of ppUL44 homologues from different betaherpesviruses, including human herpesvirus 6 (HHV-6) and 7 (HHV-7), showed that all possess similar sequences in the same position (44) (Fig. ​(Fig.1B),1B), implying functional significance.Open in a separate windowFIG. 1.The highly conserved flexible loop (residues 162 to 174) within ppUL44 protrudes from ppUL44 basic face and is important for efficient nuclear accumulation and localization in nuclear speckles. (A) Schematic representation of ppUL44 N-terminal domain (residues 9 to 270, protein data bank accession no. 1T6L) generated using the Chimera software based on the published crystal structure (10, 35). Color: yellow, β-sheets; red, α-helices. Residues involved in ppUL44 dimerization (P85, L86, L87, L93, F121, and M123), as well as basic residues potentially involved in DNA binding (K21, R28, K32, K35, K128, K158, K224, and K237), are represented as spacefill in orange and green, respectively. Residues P162 and C175, in black, are indicated by arrowheads, while residues 163 to 174 are not visible in the electron density maps and could potentially extend in the cavity formed by ppUL44''s basic face to directly contact DNA. Residues forming ppUL44 connector loop (128-142) are in blue. (B) Sequence alignment between HCMVUL44-FL and the corresponding region of several betaherpesvirus ppUL44 homologues. The single-letter amino acid code is used, with basic residues in boldface. (C) COS-7 cells were transfected to express the indicated GFP fusion proteins and imaged live 16 h after transfection using CLSM and a 40× water immersion objective lens. (D) Quantitative results for the Fn/c and speckle formation for GFP-UL44 fusion proteins. The data for the Fn/c ratios represent the mean Fn/c relative to each protein indicated as a percentage of the mean Fn/c relative to GFP-UL44wt ± the standard error of the mean, with the number of analyzed cells in parentheses. (E) HEK 293 cells expressing the indicated GFP-UL44 fusion proteins were lysed, separated by PAGE, and analyzed by Western blotting as described in Materials and Methods, using either the anti-GFP or the anti-α-tubulin MAbs.A recent study revealed that substitution of UL44-FL basic residues with alanine residues strongly impairs the ability of a bacterially expressed N-terminal fragment of UL44 to bind 30-bp dsDNA oligonucleotides in vitro, suggesting that UL44-FL could be involved in dsDNA-binding during viral replication (22). However, the role of UL44-FL in mediating the binding of full-length UL44 to dsDNA in cells and its role in DNA replication have not been investigated. We use here a variety of approaches to delineate the role of UL44-FL in living cells, our data revealing that UL44-FL is not required for ppUL44 dimerization or binding to the catalytic subunit pUL54 but is crucial for HCMV oriLyt-dependent DNA replication, being required for the formation of nuclear aggregates, nuclear accumulation/retention, and DNA binding of ppUL44. Importantly, ppUL44Δloop exhibits a transdominant-negative phenotype, inhibiting HCMV oriLyt-dependent DNA replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for dsDNA binding. This underlines ppUL44-FL as an important determinant for HCMV replication in a cellular context for the first time, with potential implications for the development of novel antiviral approaches.     

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.