Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

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).     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup

Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault''s rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault''s rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault''s rousette.Coronaviruses infect a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterization, coronaviruses were traditionally classified into three distinct groups, groups 1, 2, and 3 (3, 27, 59). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has renamed the traditional group 1, 2, and 3 coronaviruses as Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx). Coronaviruses are known to have a high frequency of recombination as a result of their unique mechanism of viral replication (27). Such tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (24, 47, 52).The severe acute respiratory syndrome (SARS) epidemic has boosted interest in the study of coronaviruses in humans and animals (21, 34, 38, 41, 54). In the past few years, there has been a dramatic increase in the number of newly described human and animal coronaviruses (2, 4, 5, 8-10, 15-20, 23, 25, 28, 30, 32, 35, 36, 39, 43, 45, 50, 51, 53, 56, 58). Two novel human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1), belonging to Alphacoronavirus and Betacoronavirus, respectively, have been discovered, in addition to the human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), and SARS coronavirus (SARS-CoV) (17, 29, 45, 53, 55). We have also previously described the discovery of a diversity of novel coronaviruses in wild bats and birds in China, including SARSr-Rh-BatCoV, belonging to Betacoronavirus subgroup B, from Chinese horseshoe bats (30, 48, 56). Among these novel coronaviruses, three avian coronaviruses were found to belong to a novel subgroup of Gammacoronavirus (Gammacoronavirus subgroup C), while three bat coronaviruses were found to belong to two novel subgroups of Betacoronavirus (Betacoronavirus subgroups C and D) (48, 50). Based on the presence of the huge diversity of coronaviruses in Alphacoronavirus and Betacoronavirus among various bat species, bats are likely the reservoir for the ancestor of these two coronavirus genera (47).During our genome analysis of these novel coronaviruses, one of them, Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), belonging to Betacoronavirus subgroup D, which was identified in Leschenault''s rousette bats, was found to display marked nucleotide and amino acid sequence polymorphism among the four strains with complete genome sequences (50). In our study on HCoV-HKU1, it has been shown that such sequence polymorphisms may indicate the presence of different genotypes (52). By complete genome sequence analysis of the potentially different genotypes of HCoV-HKU1, we have demonstrated for the first time natural recombination in a human coronavirus, resulting in the generation of at least three genotypes (52). We have also recently shown that recombination between different strains of SARSr-Rh-BatCoV from different regions of China may have given rise to the emergence of civet SARSr-CoV (31). To investigate the presence of different genotypes of Ro-BatCoV HKU9, the complete RNA-dependent RNA polymerase (RdRp) (corresponding to nsp12), spike (S), and nucleocapsid (N) gene sequences of Ro-BatCoV HKU9 from 10 additional bats were determined. Since sequence analysis showed the possible coexistence of different genotypes in seven bat individuals, complete genome sequencing of these distinct genotypes from two bats was carried out to investigate for possible recombination events among the different genotypes. In addition, serological characterization of Ro-BatCoV HKU9 was also performed by Western blot and enzyme immunoassays using recombinant Ro-BatCoV HKU9 nucleocapsid proteins and recombinant nucleocapsid proteins of Betacoronavirus subgroup A, B, and C coronaviruses to determine possible cross-reactivity among the different Betacoronavirus subgroups and the seroepidemiology of Ro-BatCoV HKU9 in Leschenault''s rousette bats.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.