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.     

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.     

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).     

Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.Our understanding of herpesvirus morphogenesis is mainly derived from studies of Alphaherpesvirinae, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV). A faster replication cycle and a more productive infection in tissue culture aided genetic analysis of alphaherpesvirus morphogenesis. In addition, deletion mutants of key morphogenesis genes in alphaherpesviruses often maintain basic replication capacity, whereas the mutations of their homologues in Betaherpesvirinae or Gammaherpesvirinae mostly result in a lethal phenotype (for the UL31 and the UL34 family, see references 3, 6, 9-11, 16, 20, 21, and 42). These genes became amenable to comprehensive genetic analysis in betaherpesviruses only after their genomes were cloned as infectious bacterial artificial chromosomes (BACs), which obviated the need to generate replication-competent intermediates or complementing cell lines (3, 21, 23). BAC-based mutagenesis allowed viability screens mapping essential genes (8, 43) or even functional sites of essential genes in cytomegaloviruses (3, 21). However, these approaches cannot easily be applied to reveal the null phenotypes in the context of virus replication, as mutant viruses are not easily reconstituted. In addition, deletion of an essential viral gene can reveal the null phenotype of only the first of perhaps several essential functions during virus morphogenesis. This problem can be addressed to some extent by using dominant negative (DN) mutations (36). DN mutants are loss-of-function mutants that induce a null phenotype in the presence of the wild-type (wt) allele (14). Analysis of phenotypes induced by DN mutants proved to be extremely useful in genetics and cell biology, signaling, and biochemistry. Such inhibitory mutants of cellular proteins are often designed based on knowledge on the structural or functional role of a well-characterized protein domain. Unfortunately, we lack the structural information that would allow knowledge-based design of viral DN mutants for the majority of herpesvirus gene products. Thus, we established a random screen consisting of three steps to identify mutants of viral genes with DN potential (36): (i) a library of mutants is generated by random insertion of 5 amino acids (aa) or a stop codon into the open reading frame (ORF) of interest using transposon mutagenesis, (ii) nonfunctional mutants are identified by cis complementation of the respective deletion mutant mouse cytomegalovirus (MCMV) BAC, and (iii) nonfunctional mutants are tested for their inhibitory potential upon reconstitution of the wt BAC cloned genomes. In the last screen, mutants that have a specific inhibitory effect on the activity of the wt allele are selected. The specific phenotype obtained upon induction of the inhibitory mutants in the context of virus replication is then verified and further characterized using a tetracycline (Tet) regulon-based viral conditional expression system (36, 37).One intriguing aspect of herpesvirus morphogenesis is the transition of capsids from the nuclear to the cytoplasmic phase of virus morphogenesis. Two conserved nonstructural proteins, the homologues of the membrane protein pUL34 and its nuclear partner protein pUL31, form a nuclear egress complex (NEC) (18, 27, 42), which is required for primary envelopment and export of nuclear capsids to the cytoplasm (reviewed in references 24 and 25). Recent studies have revealed that the homologues of alphaherpesvirus pUL34 and pUL31, the M50 and the M53 gene products of the betaherpesvirus MCMV (pM50 and pM53, respectively) and the BFRF1 and the BFLF2 gene products of the gammaherpesvirus Epstein-Barr virus (EBV), apparently share the major functions of these two proteins. The lack of one or both proteins of the NEC generally results in the retention of viral capsids in the nucleus. This is lethal for beta- and gammaherpesvirus production (3, 9-11, 16, 18, 21, 27, 35, 42).The details of the mechanisms by which the NEC proteins mediate capsid export through the nuclear envelope are poorly understood. We (3, 21, 36, 38) and others (1, 19, 34) have started to dissect details of the NEC function using a genetic approach based on subtle mutagenesis of the respective genes. Analysis of the MCMV M50 gene by comprehensive mutagenesis localized two different functional sites. They were the M53 binding site within the N-terminal domain of M50, as well as the transmembrane region at its C terminus (3). Liang and Baines located the respective binding site in HSV-1 UL34 at aa 137 to 181 (19). Our approach, based on screens for DN mutants, identified a proline-rich sequence (aa 179 to 207) in the M50 gene product as an additional essential region (36). A recombinant virus expressing an M50 mutant lacking this site was defective in capsid egress from the nucleus despite the presence of the wt M50 protein. Consequently, the production of infectious particles after infection was reduced by more than 2 orders of magnitude. The UL34 homologues of alpha- and gammaherpesviruses lack a similar polyproline motif, but the result was confirmed by mutating the human cytomegalovirus (HCMV) homologue UL50 at the corresponding region, which is conserved within betaherpesviruses (36). The M50 mutants lacking the proline-rich motif still bind and colocalize to their respective NEC partner, pM53. Interestingly, Bjerke and coworkers also provided genetic evidence for the existence of at least one additional, yet-unknown, but essential functional entity in pUL34 of HSV-1, besides its known pUL31 binding activity, using a screen based on charged-cluster mutations (1). Further analysis of one of the noncomplementing charged-cluster mutants carrying the defect in the N-terminal domain of pUL34 also revealed a DN activity and suggested a new functional site involved in membrane curvature formation, together with the C-terminal domain of UL31 (34).The genetic analysis of M53 by Tn7-based linker scanning mutagenesis, followed by a cis complementation assay, localized the M50-binding site between aa 112 and 137 within the first of the four conserved regions (CRs) shared among the herpesvirus UL31 homologues (21). This analysis, together with a study we performed for further characterization of pM50/pM53 interaction, revealed that the large C-terminal part of pM53, comprising CR2 to -4, must carry at least one additional, yet-unknown, but essential functional site (21, 38).Here, we screened loss-of-function mutants of the MCMV M53 gene to retrieve M53 alleles with DN activity to localize this new functional domain. Mutants with a very strong inhibitory potential accumulated within CR4 of pM53 close to its C terminus. These CR4 mutants induced a block of capsid export from the nucleus. In addition, we could associate these mutations with the induction of a defect in capsid maturation and/or DNA packaging. These data suggested that pM53 is not only crucial for nuclear egress, but also involved in earlier steps of MCMV morphogenesis.     

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.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.