Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.Primate immunodeficiency viruses infect helper T lymphocytes and cells of the macrophage/monocyte lineage by binding of their viral envelope glycoprotein, Env, to a combination of two host cell-specific surface proteins, CD4 and either the CCR5 or CXCR4 chemokine receptors (reviewed in reference 62). Ensuing fusion of the viral envelope with the host cell plasma membrane delivers the viral genetic material into the cytoplasm. Remarkably, the most highly transcribed viral gene in the early phase of infection does not encode an enzyme or structural protein but an accessory protein named Nef. Early expression of Nef is thought to reprogram the host cell for optimal replication of the virus. Indeed, Nef has been shown to enhance virus production (19, 24, 59, 74) and to promote progression to AIDS (23, 47, 48), making it an attractive candidate for pharmacologic intervention.Nef is an N-terminally myristoylated protein with a molecular mass of 27 kDa for human immunodeficiency virus type 1 (HIV-1) and 35 kDa for HIV-2 and simian immunodeficiency virus (27, 29, 50, 65). Nef has been ascribed many functions, the best characterized of which is the downregulation of the CD4 coreceptor from the surface of infected cells (28, 35, 57). CD4 downregulation is believed to prevent superinfection (8, 52) and to preclude the cellular retention of newly synthesized Env (8, 49), thus allowing the establishment of a robust infection (30, 71).The molecular mechanism by which Nef downregulates CD4 has been extensively studied. A consensus has emerged that Nef accelerates the endocytosis of cell surface CD4 (2, 64) by linking the cytosolic tail of CD4 to the heterotetrameric (α-β2-μ2-σ2) adaptor protein-2 (AP-2) complex (17, 25, 34, 45, 67). Determinants in the CD4 tail bind to a hydrophobic pocket comprising tryptophan-57 and leucine-58 on the folded core domain of Nef (34). On the other hand, a dileucine motif (i.e., ENTSLL, residues 160 to 165) (14, 22, 32) and a diacidic motif (i.e., DD, residues 174 and 175) (3) (residues correspond to the NL4-3 clone of HIV-1) within a C-terminal, flexible loop of Nef bind to the α and σ2 subunits of AP-2 (17, 18, 25, 51). AP-2, in turn, binds to clathrin, leading to the concentration of CD4 within clathrin-coated pits (15, 33). These pits eventually bud from the plasma membrane as clathrin-coated vesicles that deliver internalized CD4 to endosomes. In essence, then, Nef acts as a connector that confers on CD4 the ability to be rapidly internalized in a manner similar to endocytic receptors (75).Unlike typical endocytic recycling receptors like the transferrin receptor or the low-density lipoprotein receptor, however, CD4 that is forcibly internalized by Nef does not return to the cell surface but is delivered to lysosomes for degradation (4, 64, 68). Thus, expression of Nef decreases both the surface and total levels of CD4. What keeps internalized CD4 from returning to the plasma membrane? We hypothesized that Nef might additionally act on endosomes to direct CD4 to lysosomes. This is precisely the fate followed by signaling receptors, transporters, and other transmembrane proteins that undergo ubiquitination-mediated internalization and targeting to the multivesicular body (MVB) pathway (40, 46). This targeting involves the endosomal sorting complex required for transport (ESCRT), including the ESCRT-0, -I, -II, and -III complexes, which function to sort ubiquitinated cargoes into intraluminal vesicles of MVBs for eventual degradation in lysosomes (40, 46). Herein, we show that Nef indeed plays a novel role in targeting internalized CD4 from endosomes to the MVB pathway in an ESCRT-dependent manner. We also show that both Nef and CD4 undergo ubiquitination on lysine residues, but, strikingly, this modification is not required for CD4 targeting to the MVB pathway.     

Poxvirus Complement Control Proteins Are Expressed on the Cell Surface through an Intermolecular Disulfide Bridge with the Viral A56 Protein

The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.Poxviruses, including vaccinia virus (VACV), encode large numbers of immunomodulatory proteins that help them establish an infection and combat the host''s immune response (10, 32). One of these is the vaccinia virus complement control protein (VCP), which is both secreted from and expressed on the surface of infected cells (9, 14, 16, 17). VCP acts against the complement system, a series of soluble proteins that is an important early component of the innate immune system and also shapes adaptive immune responses (15, 42, 43). In response to viral infection, complement can opsonize or inactivate virions and can lyse enveloped virus or infected cells (1, 3, 7, 12). Because of these pressures, a number of viruses, including herpes simplex virus, flaviviruses, and poxviruses, encode novel or host-derived regulators of complement, while others, including HIV and poxviruses, incorporate host complement regulatory proteins into virus particles (7, 11, 31, 39). Many orthopoxviruses encode a complement regulator (8, 20, 23, 29), and the most studied of these is VCP. Structurally, VCP is made up of four short consensus repeats (SCR) that are the basic units of mammalian complement regulators (17, 25), and VCP has been shown to interfere with the complement cascade at multiple steps (2, 16, 20-22, 25, 28-30, 33). Additionally, a VCP knockout virus generates smaller lesions in animal models (14, 16). While some host complement control proteins (CCPs) are secreted, many contain transmembrane domains (or a glycophosphatidylinositol anchor) and are thus expressed on the cell surface (42, 43). Thus, when we found that VCP is also expressed on the infected cell surface and protects infected cells from complement-mediated lysis in vitro (9), we believed this to be an important interaction that required further investigation. We previously found that the N-terminal cysteine on VCP was needed for surface expression and that the VACV transmembrane protein A56 was also required (9). The vaccinia virus A56 protein is a type 1 transmembrane glycoprotein that is found on the surface of infected cells and on extracellular virus particles (4, 18, 26, 27, 36). It interacts with another viral protein, K2 (19, 37, 45), which lacks a transmembrane domain and binds to A56 noncovalently (36). The A56/K2 complex prevents syncytium formation between infected cells and superinfection by interacting with the vaccinia virus entry/fusion complex on virions (24, 38, 40, 41). Here we provide evidence that the N-terminal cysteine on VCP forms an intermolecular disulfide bond with cysteine 162 on the ectodomain of A56. We also demonstrate that similar interactions can occur with other poxvirus CCPs, as the smallpox virus and ectromelia virus homologs of VCP also exhibit A56-dependent surface expression.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

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

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

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.     

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

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).     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.