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1.
Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.In recent years there has been growing interest in viruses infecting hosts in the domain Archaea (43). Archaeal viruses were discovered 35 years ago (52), and today about 50 such viruses are known (43). They represent highly diverse virion morphotypes in contrast to the vast majority (96%) of head-tail virions among the over 5,000 described bacterial viruses (1). Although archaea are widespread in both moderate and extreme environments (13), viruses have been isolated only for halophiles and anaerobic methanogenes of the kingdom Euryarchaeota and hyperthermophiles of the kingdom Crenarchaeota (43).In addition to soil and marine environments, high viral abundance has also been detected in hypersaline habitats such as salterns (i.e., a multipond system where seawater is evaporated for the production of salt) (19, 37, 50). Archaea are dominant organisms at extreme salinities (36), and about 20 haloarchaeal viruses have been isolated to date (43). The majority of these are head-tail viruses, whereas electron microscopic (EM) studies of highly saline environments indicate that the two other described morphotypes, spindle-shaped and round particles, are the most abundant ones (19, 37, 43). Thus far, the morphological diversity of the isolated haloarchaeal viruses is restricted compared to viruses infecting hyperthermophilic archaea, which are classified into seven viral families (43).All of the previously described archaeal viruses have a double-stranded DNA (dsDNA) genome (44). However, a newly characterized haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), has a single-stranded DNA (ssDNA) genome (39). HRPV-1 and its host Halorubrum sp. were isolated from an Italian (Trapani, Sicily) solar saltern. Most of the studied haloarchaeal viruses lyse their host cells, but persistent infections are also typical (40, 44). HRPV-1 is a nonlytic virus that persists in the host cells. In liquid propagation, nonsynchronous infection cycles of HRPV-1 lead to continuous virus production until the growth of the host ceases, resulting in high virus titers in the growth medium (39).The pleomorphic virion of HRPV-1 represents a novel archaeal virus morphotype constituted of lipids and two major structural proteins VP3 (11 kDa) and VP4 (65 kDa). The genome of HRPV-1 is a circular ssDNA molecule (7,048 nucleotides [nt]) containing nine putative open reading frames (ORFs). Three of them are confirmed to encode structural proteins VP3, VP4, and VP8, which is a putative ATPase (39). The ORFs of the HRPV-1 genome show significant similarity, at the amino acid level, to the minimal replicon of plasmid pHK2 of Haloferax sp. (20, 39). Furthermore, an ∼4-kb region, encoding VP4- and VP8-like proteins, is found in the genomes of two haloarchaea, Haloarcula marismortui and Natronomonas pharaonis, and in the linear dsDNA genome (16 kb) of spindle-shaped haloarchaeal virus His2 (39). The possible relationship between ssDNA virus HRPV-1 and dsDNA virus His2 challenges the classification of viruses, which is based on the genome type among other criteria (15, 39).HRPV-1 is proposed to represent a new lineage of pleomorphic enveloped viruses (39). A putative representative of this lineage among bacterial viruses might be L172 of Acholeplasma laidlawii (14). The enveloped virion of L172 is pleomorphic, and the virus has a circular ssDNA genome (14 kb). In addition, the structural protein pattern of L172 with two major structural proteins, of 15 and 53 kDa, resembles that of HRPV-1.The structural approach has made it possible to reveal relationships between viruses where no sequence similarity can be detected. It has been realized that several icosahedral viruses infecting hosts in different domains of life share common virion architectures and folds of their major capsid proteins. These findings have consequences for the concept of the origin of viruses. A viral lineage hypothesis predicts that viruses within the same lineage may have a common ancestor that existed before the separation of the cellular domains of life (3, 5, 8, 26). Currently, limited information is available on the detailed structures of viruses infecting archaea. For example, the virion structures of nontailed icosahedral Sulfolobus turreted icosahedral virus (STIV) and SH1 have been determined (21, 23, 46). However, most archaeal viruses represent unusual, sometimes nonregular, morphotypes (43), which makes it difficult to apply structural methods that are based on averaging techniques.A biochemical approach, i.e., controlled virion dissociation, gives information on the localization and interaction of virion components. In the present study, controlled dissociation was used to address the virion architecture of HRPV-1. A comparative lipid analysis of HRPV-1 and its host was also carried out. Our results show that the unique virion type is composed of a flexible membrane decorated with the glycosylated spikes of VP4 and internal membrane protein VP3. The circular ssDNA genome resides inside the viral membrane vesicle without detected association to any nucleoproteins.  相似文献   

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UL31 and UL34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pUL31, pUL34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a UL34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the UL34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on UL34 (P < 0.0001). pUL34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pUL34/gD interaction in recruiting gD to the NM. The effects of UL34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pUL31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, UL31 and UL34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.Herpesvirus virions comprise a nucleocapsid containing genomic viral DNA, a proteinaceous tegument layer surrounding the nucleocapsid, and a virion envelope surrounding the tegument. The envelope of extracellular herpes simplex virus (HSV) virions contains glycoproteins gB, gC, gD, gE, gI, gG, gH, gK, gL, and gM (23, 51).As viewed by electron microscopy, nascent virions form as the nucleocapsid buds through densely staining regions of the nuclear membrane (NM) (21, 41). Electron tomograms of HSV perinuclear virions compared to those of extracellular virions infer that the former contain glycoproteins of considerably less glycosylation and a relatively sparse tegument layer compared to their counterparts in mature extracellular virions (6). The lower levels of glycosylation in HSV perinuclear virions are consistent with the fact that the lumen of the perinuclear space is continuous with that of the endoplasmic reticulum. Thus, the polysaccharide moieties of virion glycoproteins become fully processed as virions access Golgi enzymes during their egress to the extracellular space. Although the full proteome of the nascent perinuclear virion is unknown, immunogold studies have shown that they contain at least pUL31, pUL34, pUS3, gB, gC, gD, gH, gM, and the VP16 and pUL11 tegument proteins in addition to the proteins that comprise the viral capsid (4, 5, 15, 25, 37, 40, 47, 50, 55).The UL31 and UL34 gene products of HSV-1 (pUL31 and pUL34, respectively) form a complex that localizes at the inner and outer NMs (INM and ONM, respectively) of infected cells (40). Both proteins are essential for nucleocapsid envelopment at the INM and become incorporated into nascent virions when nucleocapsids bud through the INM into the perinuclear space (39, 40, 42). The proteins and their essential role in nucleocapsid envelopment are conserved in all herpesvirus subfamilies (14, 20, 32, 45). pUL31 of HSV-1 is a mostly hydrophobic phosphoprotein that is held in close approximation to the nucleoplasmic face of the INM by interaction with pUL34, an integral membrane protein of type II orientation (33, 40, 46, 56). The first 248 amino acids of pUL34 are predicted to reside in the nucleoplasm or cytoplasm, depending on whether the protein localizes in the INM or ONM, respectively. This is followed by an approximately 22-amino acid transmembrane domain with up to 5 amino acids residing in the perinuclear space or lumen of the endoplasmic reticulum.In the most prominent model of herpesvirion egress, the envelope of the perinuclear virion fuses with the ONM, releasing the deenveloped nucleocapsid into the cytoplasm, where it subsequently buds into cytoplasmic membranous organelles such as the Golgi or trans-Golgi network (34, 49). This model is supported by the observation that pUL31 and pUL34 are located in the perinuclear virion but not extracellular virions (18, 40). Thus, these proteins are lost from the virion upon fusion of the virion envelope with the ONM. Also supporting this egress model is the observation that deletion of both gB and gH causes virions to accumulate aberrantly in the perinuclear space (15). The involvement of gH and gB is potentially satisfying because these proteins comprise essential components of the machinery that mediates fusion of the virion envelope with the plasma or endosomal membranes during the initiation of infection (9, 12, 16, 44, 52). Moreover, expression of a combination of gB, gD, gH, and gL is sufficient to mediate fusion of cell membranes, whereas coexpression with gM or gK inhibits this fusion (3, 8, 11). Although the mechanism of fusion is unclear, gD is known to bind viral receptors on cell surfaces, and the structure of gB indicates features reminiscent of other viral fusion proteins (24, 35, 48). gD has been shown to interact with gB and gH at least transiently, suggesting that these interactions may be important for the fusion reaction (1, 2). Thus, fusion between the nascent and mature virion envelopes with target membranes may share mechanistic similarities.On the other hand, it is likely that the two fusion events are mechanistically distinct because (i) single deletion of either gH or gB precludes viral entry and cell/cell fusion but does not cause nascent virions to accumulate in the perinuclear space (9, 16, 31, 43) and (ii) the activity of a viral kinase encoded by US3 is dispensable for entry but believed to promote fusion of the perinuclear virion and ONM (28, 40). Moreover, the lack of glycoproteins from the pseudorabies virus perinuclear virion suggests that fusion is mediated by an entirely different mechanism in this system (26).The current study focuses on how glycoproteins are incorporated into the nascent virion. We show that optimal recruitment of gD to both leaflets of the NM and gM to the INM requires pUL34 and pUL31. We also show that immature gD interacts with pUL34, suggesting a mechanism by which pUL34 might recruit gD to the NM.  相似文献   

4.
Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3′ leader and 5′ trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3′ untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.Outbreaks of infections with many novel paramyxoviruses causing catastrophic illnesses have been reported all over the world in the last few decades. A large number of diverse host species have been involved, including avian, porcine, canine, bovine, equine, ovine, human, reptilian, and aquatic species (22, 29, 40, 50, 51). Cases of cross-species transmission and pathogen jumping to humans were also reported (10, 20), demonstrating the value of characterizing new animal pathogens, even if their pathogenic potential is currently unknown. Prior to the 1990s, only La Piedad Michoacán paramyxovirus had been well studied as a neurotropic paramyxovirus isolated from pigs. Many paramyxovirus porcine pathogens have been reported since the 1950s in numerous countries, including Japan (55), Canada (18), and Israel (32), as well as the United States (25, 32). There was also a case of concurrent infection with a porcine reproductive and respiratory syndrome virus and a paramyxovirus which was subsequently named SER virus (70) in Germany in the 1990s (28). Four bat-associated paramyxoviruses were reported to cause disease in animals and humans in 1994 (72). Hendra virus and Nipah virus, which caused severe respiratory disease and death in horses and their trainer and severe febrile encephalitis and death in pigs and farmers, respectively, have been classified as members of the genus Henipavirus in the subfamily Paramyxovirinae (7, 9, 20, 30, 48). Some recently isolated viruses, such as Menangle virus (55), Tupaia paramyxovirus (69), Tioman virus (11), Mossman virus (47), J-virus (31, 33), Beilong virus (42), Mapuera virus (34), Tursiops truncatus parainfluenzavirus 1 (PIV1), isolated from bottlenose dolphins (50), and Atlantic salmon paramyxovirus (51), remain unclassified below the subfamily level. All members of the subfamily Paramyxovirinae have six genes in the following order: 3′-N-P-M-F-A-L-5′, where N, P, M, F, A, and L indicate the genes for the nucleocapsid protein, the phosphoprotein, and the matrix, fusion, attachment, and large polymerase proteins, respectively (40).Recently, we reported the antigenic and molecular characterization of glycoprotein genes from two novel swine PIV3 (sPIV3) isolates from the brains of pigs that experienced respiratory and central nervous system disease (57). These two sPIV3 strains were antigenically and genetically very closely related to bovine PIV3 (bPIV3) in the genus Respirovirus (57). However, the pathogenicity of these sPIV3 strains in conventionally reared pigs and the complete genome sequences of these isolates are presently unknown.In bovines, bPIV3 infection results in asymptomatic to severe respiratory disease, but no neurological disease has been reported (16). Limited sequence polymorphism among the bPIV3 strains was detected previously (12, 66). Recently, after an analysis of Australian isolates of bPIV3, two distinct genotypes of bPIV3, A and B, were proposed (29). In this study, we have performed a complete genome sequence analysis of these swine isolates and determined their pathogenicity in conventionally reared pigs. Our analysis indicated that there are two distinct genetic groupings discernible within genotype A, represented by bPIV3 shipping fever strain (bPIV3-SF)-like and bPIV3 strain 910N (bPIV3-910N)-like viruses, with one swine isolate in each of these groups. Several amino acid residues that may reflect the minor population variations in the new host due to cross-species infection were identified. But both swine viruses induced a very mild respiratory illness without any neurological signs in young piglets, suggesting that coinfection with other infectious agents or the presence of other environmental factors may be required to precipitate clinical disease.  相似文献   

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Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3Dpol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.  相似文献   

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We have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an ∼17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Haloarcula provirus), and the Haloarcula virus SH1. These genetic elements share two major capsid proteins and a putative packaging ATPase. The ATPase is similar with the ATPases found in the PRD1-type viruses, thus providing an evolutionary link to these viruses and furthering our knowledge on the origin of viruses.Three-dimensional structures of the major capsid proteins, as well as the architecture of the virion and the sequence similarity of putative genome packaging ATPases, have revealed unexpected evolutionary connection between virus families. Viruses infecting hosts residing in different domains of life (Bacteria, Archaea, and Eukarya) share common structural elements and possibly also ways to package the viral genome (8, 13, 41). It has been proposed that the set of genes responsible for virion assembly is a hallmark of the virus and is designated as the innate viral “self,” which may retain its identity through evolutionary times (5). Based on this, it is proposed that viruses can be classified into lineages that span the different domains of life. Therefore, the studies of new virus isolates might provide insights into the events that led to the origin of viruses and maybe even the origin of life itself (34, 40). However, viruses are known to be genetic mosaics (28), and these structural lineages therefore do not reflect the evolutionary history of all genes in a given virus. For example, the genome replication strategies vary significantly even in the currently established lineages (41) and, consequently, a structural approach does not point out to a specific form of replication in the ancestor. Nevertheless, as the proposal for a viral self is driven from information on viral structures and pathways of genome encapsidation, the ancestral form of the self was likely to be composed of a protective coat and the necessary mechanisms to incorporate the genetic material within the coat.Viruses structurally related to bacteriophage PRD1, a phage infecting gram-negative bacteria, have been identified in all three domains of life, and the lineage hypothesis was first proposed based on structural information on such viruses. Initially, PRD1 and human adenovirus were proposed to originate from a common ancestor mainly due to the same capsid organization (T=25) and the major coat protein topology, the trimeric double β-barrel fold (12). In addition, these viruses share a common vertex organization and replication mechanism (20, 31, 53, 63). PRD1 is an icosahedral virus with an inner membrane, whereas adenovirus lacks the membrane. Later, many viruses with similar double β-barrel fold in the major coat protein have been discovered and included to this viral lineage. For example, the fold is present in Paramecium bursaria Chlorella virus 1 (56) of algae, Bam35 (45) of gram-positive bacteria, PM2 (2) of gram-negative marine bacteria, and Sulfolobus turreted icosahedral virus (STIV) (38) of an archaeal host. Moreover, genomic analyses have revealed a common set of genes in a number of nucleocytoplasmic large DNA viruses. Chilo iridescent virus and African swine fever virus 1 are related to Paramecium bursaria Chlorella virus 1 and most probably share structural similarity to PRD1-type viruses (13, 30, 31, 68). The largest known viruses, represented by mimivirus and poxvirus, may also belong to this lineage (29, 77). Two euryarchaeal proviruses, TKV4 and MVV, are also proposed to belong to this lineage based on bioinformatic searches (42). The proposed PRD1-related viruses share the same basic architectural principles despite major differences in the host organisms and particle and genome sizes (1, 2, 38, 56). PM2, for example, has a genome of only 10 kbp, whereas mimivirus (infecting Acanthamoeba polyphaga) double-stranded DNA (dsDNA) genome is 1.2 Mbp in size (59).How many virion structure-based lineages might there be? This obviously relates to the number of protein folds that have the properties needed to make viral capsids. It has been noted that, in addition to PRD1-type viruses, at least tailed bacterial and archaeal viruses, as well as herpesviruses, share the same coat protein fold. Also, certain dsRNA viruses seem to have structural and functional similarities, although their hosts include bacteria and yeasts, as well as plants and animals (6, 18, 19, 27, 55, 60, 74). Obviously, many structural principles to build a virus capsid exist, and it has been suggested that especially geothermally heated environments have preserved many of the anciently formed virus morphotypes (35).Thermophilic dsDNA bacteriophage P23-77 was isolated from an alkaline hot spring in New Zealand on Thermus thermophilus (17) ATCC 33923 (deposited as Thermus flavus). P23-77 was shown to have an icosahedral capsid and possibly an internal membrane but no tail (81). Previously, another Thermus virus, IN93, with a similar morphology has been described (50). IN93 was inducible from a lysogenic strain of Thermus aquaticus TZ2, which was isolated from hot spring soil in Japan. Recently, P23-77 was characterized in more detail (33). It has an icosahedral protein coat, organized in a T=28 capsid lattice (21). The presence of an internal membrane was confirmed, and lipids were shown to be constituents of the virion. Ten structural proteins were identified, with apparent molecular masses ranging from 8 to 35 kDa. Two major protein species with molecular masses of 20 and 35 kDa were proposed to make the capsomers, one forming the hexagonal building blocks and the other the two towers that decorate the capsomer bases (33). Surprisingly, P23-77 is structurally closest to the haloarchaeal virus SH1, which is the only other example of a T=28 virion architecture (32, 33). In both cases it was proposed that the capsomers are made of six single β-barrels opposing the situation with the other structurally related viruses where the hexagonal capsomers are made of three double β-barrel coat protein monomers (8).In the present study we analyze the dsDNA genome of P23-77. Viral membrane proteins and those associated with the capsid were identified by virion dissociation studies. The protein chemistry data and genome annotation are consistent with the results of the disruption studies. A detailed analysis of the lipid composition of P23-77 and its T. thermophilus host was carried out. The data collected here reveal additional challenges in attempts to generate viral lineages based on the structural and architectural properties of the virion.  相似文献   

8.
The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M''s ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lower-respiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13, 16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32, 40, 48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11, 12, 24, 34, 36, 39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin β family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8, 9, 18, 19, 37, 42, 43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recognized NES, appears to be responsible for its export from the nucleus, bound to the influenza virus RNA (3).We have recently shown that RSV M localizes in the nucleus through a conventional nuclear import pathway dependent on the nuclear import receptor importin β1 (IMPβ1) and the guanine nucleotide-binding protein Ran (14). In the present study, we show for the first time that RSV M possesses a Crm1-dependent nuclear export pathway, based on experiments using the specific inhibitor leptomycin B (LMB) (25), both in RSV-infected cells and in green fluorescent protein (GFP)-M fusion protein-expressing transfected cells. We use truncated and point-mutated M derivatives to map the Crm1-recognized NES within the M sequence and show that Crm1-dependent nuclear export is critical to the RSV infectious cycle, since LMB treatment early in infection, inhibiting M export from the nucleus, reduces RSV virion production and a recombinant RSV carrying a NES mutation in M was unable to replicate, probably because M deficient in nuclear export could not localize to areas of virus assembly, as shown in RSV-infected cells transfected to express GFP-M. This is the first report of a Crm1-mediated nuclear export pathway for a paramyxovirus M protein, with implications for the trafficking and function of other paramyxovirus M proteins.  相似文献   

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12.
During the search for haloarchaeal viruses, we isolated and characterized a new pleomorphic lipid-containing virus, Haloarcula hispanica pleomorphic virus 1 (HHPV-1), that infects the halophilic archaeon Haloarcula hispanica. The virus contains a circular double-stranded DNA genome of 8,082 bp in size. The organization of the genome shows remarkable synteny and amino acid sequence similarity to the genome and predicted proteins of the halovirus HRPV-1, a pleomorphic single-stranded DNA virus that infects a halophilic archaeon Halorubrum sp. Analysis of the two halovirus sequences, as well as the entire nucleotide sequence of the 10.8-kb pHK2-plasmid and a 12.6-kb chromosomal region in Haloferax volcanii, allows us to suggest a new group of closely related viruses with genomes of either single-stranded or double-stranded DNA. Currently, closely related viruses are considered to have the same genome type. Our observation clearly contradicts this categorization and indicates that we should reconsider the way we classify viruses. Our results also provide a new example of related viruses where the viral structural proteins have not diverged as much as the proteins associated with genome replication. This result further strengthens the proposal for higher-order classification to be based on virion architecture rather than on genome type or replication mechanism.Metagenomic studies have increased the amount of information on the nucleotide sequence space in our environment. It has also increased our awareness of the viral abundance and diversity not recognized before (16, 24, 26). Along with this new information, we have learned to acknowledge the significance of viruses in the evolution and behavior of other organisms (55). To reveal the dynamics and molecular interactions in the interplay between a particular virus and its host, however, isolation of single viruses and their hosts is needed. Even though a number of viruses pathogenic to humans, domestic animals, and plants, as well as some bacteriophages, have been studied in great detail, much of the diversity of the archaeal viruses has remained unknown. By the year 2007 only 44 archaeal viruses had been described (2). That embraces less than 1% of all reported viruses. Although the diversity among these few isolated archaeal viruses is considerable, a head-and-tail morphology is prevalent among isolated viruses infecting euryarchaeal cells. In contrast, viruses of Crenarchaeota are diverse and often unusual with no viruses having a head-tail morphology (53).Archaeal haloviruses infect euryarchaeal hosts living in environments up to saturated salt. This makes them an interesting group of viruses that reside in a very restricted habitat. In samples taken from high salt environments, the Dead Sea and Spanish solar salterns, viral morphotypes most often observed were spindle-shaped, head-and-tail or tailless icosahedral particles (25, 31, 47). Isolated haloviruses, however, do not seem to reflect the proportions of different morphotypes found in the nature as nearly all of the isolates possess a head-and-tail morphology (2). Molecular level studies on only two spindle-shaped (10, 11) and one tailless icosahedral particle have been carried out (37, 51). Virus-like particles of other morphologies have also been observed in high-salt environments (47), but only one additional morphotype has been described in detail (50). This recently isolated lipid containing halovirus, HRPV-1, is the first archaeal virus containing a single-stranded DNA (ssDNA) genome (50). It infects Halorubrum sp. and has a pleomorphic appearance with glycosylated spike structures protruding from its external membrane (49, 50).The evolution of prokaryotic viral genome sequences is very fast (18), and the assessment of viral relationships using homology of the genome sequences applies only to closely related viruses (17, 19). Current higher-order classification of viruses is based on the host organism, the nature of the genome (RNA/DNA, single stranded versus double stranded) and the virion morphology. Recently, a higher-order clustering of virus families has been proposed based on common principles of virion architectures as well as on the fold of the major capsid protein (1, 6, 12, 13, 42). Consequently, major capsid proteins most probably belong to the vertically inherited viral “self” (4), whereas proteins involved in replication of the viral genome can be swapped by horizontal exchange (21, 63). The proposal is based on observations that structurally related viruses have been found to infect organisms that reside in all three domains of life.We have isolated a new pleomorphic virus infecting Haloarcula hispanica (Har. hispanica pleomorphic virus 1 [HHPV-1]). Here, we determine the molecular constituents of HHPV-1 and its genetic relatedness to other archaeal viruses and putative proviruses. Sequence homology and gene order (synteny) shows distinct genomic regions shared between four genetic elements separating replication, virus assembly, and integration functions. Surprisingly, in spite of the close relatedness of HRPV-1 and HHPV-1, the genome types of these two viruses differ (ssDNA and dsDNA, respectively).  相似文献   

13.
14.
Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.Bats belong to one of the most diverse, abundant, and widely distributed group of mammals. More than 1,100 bat species belong to the order of Chiroptera, representing approximately 20% of all mammalian species (54). Most bat species feed on insects and other arthropods, while others feed on fruit nectar, bird or mammal blood, and small vertebrates such as fish, frogs, mice, and birds (30). Of the 47 species of bats reported in the United States, most of them are insectivorous (http://www.batcon.org/).Bats are considered the natural reservoir of a large variety of zoonotic viruses causing serious human diseases such as lyssaviruses, henipaviruses, severe acute respiratory syndrome coronavirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics of bats, including their genetic diversity, broad geological distribution, gregarious habits, high population density, migratory habits, and long life span (30, 58), likely endow them with the ability to host diverse viruses, some of which are also able to infect humans and other mammals (41, 63).More than 80 virus species have been isolated or detected in bats using nucleic acid-based methods (6, 38, 59, 65). Viruses that have been recently discovered in bats include astroviruses, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68). For example, it was recently reported that a newly identified adenovirus isolated from bat guano was capable of infecting various vertebrate cell lines, including those of humans, monkeys, dogs, and pigs (35). With increasing human populations in previously wild areas, contact of bats with humans and with wild and domestic animals has increased, providing greater opportunities for cross-species transmissions of potentially pathogenic bat viruses. To better understand the range of viruses carried by bats, we undertook an initial characterization of the guano viromes of several common bat species in the United States.The development of massively parallel sequencing technology makes is possible to reveal uncultured viral assemblages within biological or environmental samples (11, 28). To date, this approach has been used to characterize viruses in equine feces (7), human blood (5), tissue (14), human feces (3, 4, 15, 45, 60, 67), and human respiratory secretions (64), which in turn has facilitated the discovery of many novel viruses (18, 20, 25, 33, 47, 50). In the present study, we analyzed the viruses present in guano from several bat species in California and Texas, using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches.  相似文献   

15.
16.
Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells. To define mumps virus (MuV) proteins important for this process, viral proteins were expressed either singly or in combination in mammalian cells to produce virus-like particles (VLPs). Only the MuV matrix (M) protein when expressed by itself was capable of inducing particle release, but the quantity of these M-alone particles was very small. Efficient production of mumps VLPs occurred only when the M protein was coexpressed together with other viral proteins, with maximum production achieved upon coexpression of the viral M, nucleocapsid (NP), and fusion (F) proteins together. Electron microscopy analysis confirmed that VLPs were morphologically similar to MuV virions. The two MuV glycoproteins were not equal contributors to particle formation. The F protein was a major contributor to VLP production, while the hemagglutinin-neuraminidase protein made a smaller contribution. Evidence for the involvement of class E protein machinery in VLP budding was obtained, with mumps VLP production inhibited upon expression of dominant-negative versions of the class E proteins Vps4A and Chmp4b. Disruption of the sequence 24-FPVI-27 within the MuV M protein led to poor VLP production, consistent with findings of earlier studies of a related sequence, FPIV, important for the budding of parainfluenza virus 5. Together, these results demonstrate that different MuV structural proteins cooperate together for efficient particle production and that particle budding likely involves host class E protein machinery.Mumps virus (MuV) is a paramyxovirus from the Rubulavirus genus. Prior to mass vaccination, mumps was a very common childhood illness, with characteristic symptoms including fever, fatigue, and inflammation of the salivary glands. Less frequently, MuV infection results in serious complications including aseptic meningitis and encephalitis (22). Significant outbreaks of mumps have occurred recently in the United Kingdom (6), Canada (40), and the United States (7, 14), highlighting the continued relevance of this disease even in countries where vaccination is widespread. Like other paramyxoviruses, MuV possesses a genome that consists of single-stranded negative-sense RNA, encapsidated by a nucleocapsid (NP) protein and associated with an RNA-dependent RNA polymerase complex composed of large protein and phosphoprotein subunits. This core is linked to the virion membrane by matrix (M) protein. The outer surface of the virion is covered with glycoprotein spikes consisting of the hemagglutinin-neuraminidase (HN) protein, which binds sialic acid to allow virion attachment to cells, and fusion (F) protein, which induces viral and cellular membranes to fuse together during virus entry. Additional components of MuV include the small hydrophobic protein, which prevents infected cells from undergoing apoptosis (67), and V protein, which prevents induction of interferon-induced antiviral responses (29, 30, 62). The late steps of the MuV life cycle that allow for assembly and budding of MuV virions remain for the most part unexplored.Enveloped virus particles are formed by budding from cellular membranes at specific locations at which viral proteins, and often host factors, have assembled together. For the negative-strand RNA viruses, coordination among the different viral components during virus assembly appears to be directed by the viral matrix proteins, which have the potential to interact with the cytoplasmic tails of the viral glycoproteins and with viral ribonucleoproteins (RNPs) in the cytoplasms of infected cells. M proteins likely assemble as layers beneath the plasma membranes of infected cells and induce other viral components to gather at these locations, from which virus budding occurs (reviewed in references 49 and 57).For many viruses, it has been possible to achieve assembly and budding of particles from cells that have been transfected to produce one or more viral proteins in the absence of virus infection. These particles often resemble virions morphologically and have been termed virus-like particles (VLPs). VLP production provides a useful means for determining the individual roles of different virus proteins in particle formation, and in some cases the VLPs themselves have shown promise as vaccines (45). For most negative-strand RNA viruses, VLP formation is critically dependent on the presence of the viral matrix proteins (49). Indeed, in the cases of Newcastle disease virus (NDV) (37) and Nipah virus (11, 38), M protein expression is sufficient for highly efficient VLP production, with no apparent need for assistance from any of the other viral structural components, such as the viral glycoproteins or NP proteins. In the case of NDV, incorporation of glycoproteins and NP proteins into the budding VLPs requires specific interactions involving the M protein, but these interactions do not appear to facilitate the budding process itself (37).Although expression of viral matrix protein is sufficient for robust VLP production in the above cases, it has long been thought that additional viral components are also important for efficient budding of many negative-strand RNA viruses. For example, an important role for viral glycoproteins in virus assembly has been established based on studies with recombinant viruses that contain glycoproteins lacking their cytoplasmic tails (4, 17, 26, 34, 35, 48, 52, 66) and analyses of assembly-defective subacute sclerosing panencephalitis measles virus strains (5, 47). In fact, recent evidence suggests that for influenza virus it is the viral glycoproteins (and not viral matrix protein) that are the main drivers of virus budding (9). For other negative-strand RNA viruses, expression of viral glycoproteins together with matrix proteins in some cases significantly enhances the efficiency of VLP release. Ebola VLPs (31), Sendai VLPs (55, 56), and parainfluenza virus 5 (PIV5)-like particles (51) are all produced more efficiently in the presence of viral glycoprotein expression. Ebola virus glycoprotein in some cell types functions during virus release to inhibit the action of tetherin, a cellular protein which functions to prevent the release of enveloped virus particles from infected cells (28). In addition to the viral glycoproteins, other viral components can also enhance the production of VLPs. Production of Ebola VLPs and PIV5-like particles can be further enhanced through expression of the corresponding NP proteins (31, 51), and Sendai VLP production is enhanced through expression of Sendai virus C protein (55). Hence, for these viruses, multiple proteins cooperate with one another to achieve maximum VLP production. The extent to which particle formation actually requires this cooperation differs, however. In the case of PIV5, it is absolutely essential; expression of the M protein alone does not lead to VLP production (51). On the other hand, cooperation among viral proteins is beneficial but not strictly required for the production of Sendai or Ebola VLPs, since expression of the matrix proteins of these viruses is sufficient for VLP production (20, 55, 56, 61).The late steps of negative-strand RNA virus budding may occur in a way that is analogous to the budding of retroviruses, which employ protein-protein interaction domains called late domains to manipulate host machinery and allow release of virus particles (reviewed in references 1 and 3). Cellular factors recruited by late domains in many cases are class E proteins that are part of the vacuolar protein sorting (Vps) pathway of the cell. Indeed, disruption of the Vps pathway through expression of dominant-negative (DN) versions of the Vps4 ATPase protein blocks the budding of many retroviruses (reviewed in reference 1), as well as the budding of Ebola virus (32), Lassa fever virus (63), and PIV5 (50). However, other negative-strand RNA viruses, such as influenza virus, bud particles in ways that are not substantially affected by disruption of the cellular Vps pathway (reviewed in reference 8).Here, experiments are described which define MuV proteins important for the assembly and budding of VLPs. Using proteins derived from the 88-1961 wild-type (wt) strain of MuV, optimal production of mumps VLPs is shown to occur upon coexpression of the MuV M, F, and NP proteins together in transiently transfected mammalian cells. Evidence is also provided that supports a role for cellular class E protein machinery in the budding of mumps VLPs.  相似文献   

17.
Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.Millions of viruses and bacteria are excreted in human fecal matter (5, 17, 82), and current methods of sewage treatment do not always effectively remove these organisms (74, 76-78). The majority of treated wastewater, as well as untreated sewage, drains into the marine environment (1) and has the potential to threaten environmental (e.g., nutrients and chemicals) (45) and public (e.g., pathogen exposure via swimming and seafood consumption) (1, 24, 28, 29, 33, 44, 57, 63) health. Currently, the U.S. Environmental Protection Agency (EPA) mandates the use of bacterial indicators such as fecal coliforms and enterococci to assess water quality (75). Although monitoring of these bacteria is simple and inexpensive, it has been shown that fecal-associated bacteria are not ideal indicators of fecal pollution.Since fecal-associated bacteria are able to live in sediments in the absence of fecal pollution (18, 32, 55), their resuspension into the water column can result in false-positive results and mask correlations between their concentrations and the extent of recent fecal pollution. Another unfavorable characteristic of current bacterial indicators is their inability to predict or correlate with the presence of pathogenic viruses (25, 40, 41, 64, 80). Human-pathogenic viruses associated with feces are generally more robust than enteric bacteria and are not as easily eliminated by current methods of wastewater treatment (43, 80). For example, adenoviruses are more resilient to tertiary wastewater treatment and UV disinfection than are bacterial indicators of fecal pollution (74). Since bacterial indicators cannot accurately depict the risks to human health from fecal pollution, several studies have proposed the use of a viral indicator of wastewater contamination (35, 41, 61).While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the development of an accurate viral indicator of sewage contamination is needed for enhanced water quality monitoring. Enteric viruses (including viruses belonging to the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae) are transmitted via the fecal-oral route and are known to be abundant in raw sewage. These viruses have been used to identify fecal pollution in coastal environments throughout the world (27, 35, 39, 40, 48, 50, 56, 57, 63, 64, 67-69, 71, 80). To determine which viruses are effective indicators of fecal pollution, it is first necessary to establish a broad, baseline understanding of the many diverse groups of eukaryotic viruses in raw sewage. Several studies have identified adenoviruses, noroviruses, reoviruses, rotaviruses, and other enteroviruses (e.g., polioviruses, coxsackie viruses, and echoviruses) in raw sewage in Australia, Europe, and South Africa (30, 47, 58, 76-78). However, no broad baseline data on the presence of eukaryotic viruses in raw sewage in the United States currently exist.This study determined the presence of 10 viral groups (adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses) in raw sewage samples collected throughout the United States. All viral groups that were detected in raw sewage were then examined further to determine if they were also present in final treated wastewater effluent. These 10 viral groups were chosen because of their potential to be transmitted via the fecal-oral route, suggesting that they might be found in raw sewage. Many of these viruses (excluding adenoviruses, enteroviruses, noroviruses, reoviruses, and rotaviruses) have not been studied in sewage despite their likely presence. Picobirnaviruses have been detected in individual fecal samples (12, 70, 79, 82); however, their presence has never been analyzed in collective waste, nor have they been proposed to be potential markers of fecal pollution. This study identified potential viral indicators of fecal pollution and will have important applications to water quality monitoring programs throughout the country.  相似文献   

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19.
The combinatorial nature of genetic recombination can potentially provide organisms with immediate access to many more positions in sequence space than can be reached by mutation alone. Recombination features particularly prominently in the evolution of a diverse range of viruses. Despite rapid progress having been made in the characterization of discrete recombination events for many species, little is currently known about either gross patterns of recombination across related virus families or the underlying processes that determine genome-wide recombination breakpoint distributions observable in nature. It has been hypothesized that the networks of coevolved molecular interactions that define the epistatic architectures of virus genomes might be damaged by recombination and therefore that selection strongly influences observable recombination patterns. For recombinants to thrive in nature, it is probably important that the portions of their genomes that they have inherited from different parents work well together. Here we describe a comparative analysis of recombination breakpoint distributions within the genomes of diverse single-stranded DNA (ssDNA) virus families. We show that whereas nonrandom breakpoint distributions in ssDNA virus genomes are partially attributable to mechanistic aspects of the recombination process, there is also a significant tendency for recombination breakpoints to fall either outside or on the peripheries of genes. In particular, we found significantly fewer recombination breakpoints within structural protein genes than within other gene types. Collectively, these results imply that natural selection acting against viruses expressing recombinant proteins is a major determinant of nonrandom recombination breakpoint distributions observable in most ssDNA virus families.Genetic recombination is a ubiquitous biological process that is both central to DNA repair pathways (10, 57) and an important evolutionary mechanism. By generating novel combinations of preexisting nucleotide polymorphisms, recombination can potentially accelerate evolution by increasing the population-wide genetic diversity upon which adaptive selection relies. Recombination can paradoxically also prevent the progressive accumulation of harmful mutations within individual genomes (18, 35, 53). Whereas its ability to defend high-fitness genomes from mutational decay possibly underlies the evolutionary value of sexuality in higher organisms, in many microbial species where pseudosexual genetic exchange is permissible among even highly divergent genomes, recombination can enable access to evolutionary innovations that would otherwise be inaccessible by mutation alone.Such interspecies recombination is fairly common in many virus families (8, 17, 27, 44, 82). It is becoming clear, however, that as with mutation events, most recombination events between distantly related genomes are maladaptive (5, 13, 38, 50, 63, 80). As genetic distances between parental genomes increase, so too does the probability of fitness defects in their recombinant offspring (16, 51). The viability of recombinants is apparently largely dependent on how severely recombination disrupts coevolved intragenome interaction networks (16, 32, 51). These networks include interacting nucleotide sequences that form secondary structures, sequence-specific protein-DNA interactions, interprotein interactions, and amino acid-amino acid interactions within protein three-dimensional folds.One virus family where such interaction networks appear to have a large impact on patterns of natural interspecies recombination are the single-stranded DNA (ssDNA) geminiviruses. As with other ssDNA viruses, recombination is very common among the species of this family (62, 84). Partially conserved recombination hot and cold spots have been detected in different genera (39, 81) and are apparently caused by both differential mechanistic predispositions of genome regions to recombination and natural selection disfavoring the survival of recombinants with disrupted intragenome interaction networks (38, 51).Genome organization and rolling circle replication (RCR)—the mechanism by which geminiviruses and many other ssDNA viruses replicate (9, 67, 79; see reference 24 for a review)—seem to have a large influence on basal recombination rates in different parts of geminivirus genomes (20, 33, 39, 61, 81). To initiate RCR, virion-strand ssDNA molecules are converted by host-mediated pathways into double-stranded “replicative-form” (RF) DNAs (34, 67). Initiated by a virus-encoded replication-associated protein (Rep) at a well-defined virion-strand replication origin (v-ori), new virion strands are synthesized on the complementary strand of RF DNAs (28, 73, 74) by host DNA polymerases. Virion-strand replication is concomitant with the displacement of old virion strands, which, once complete, yields covalently closed ssDNA molecules which are either encapsidated or converted into additional RF DNAs. Genome-wide basal recombination rates in ssDNA viruses are probably strongly influenced by the specific characteristics of host DNA polymerases that enable RCR. Interruption of RCR has been implicated directly in geminivirus recombination (40) and is most likely responsible for increased basal recombination rates both within genes transcribed in the opposite direction from that of virion-strand replication (40, 71) and at the v-ori (1, 9, 20, 69, 74).Whereas most ssDNA virus families replicate via either a rolling circle mechanism (the Nanoviridae, Microviridae, and Geminiviridae) (3, 23, 24, 31, 59, 67, 74) or a related rolling hairpin mechanism (the Parvoviridae) (25, 76), among the Circoviridae only the Circovirus genus is known to use RCR (45). Although the Gyrovirus genus (the other member of the Circoviridae) and the anelloviruses (a currently unclassified ssDNA virus group) might also use RCR, it is currently unknown whether they do or not (78). Additionally, some members of the Begomovirus genus of the Geminiviridae either have a second genome component, called DNA-B, or are associated with satellite ssDNA molecules called DNA-1 and DNA-Beta, all of which also replicate by RCR (1, 47, 68).Recombination is known to occur in the parvoviruses (19, 43, 70), microviruses (66), anelloviruses (40, 46), circoviruses (11, 26, 60), nanoviruses (30), geminivirus DNA-B components, and geminivirus satellite molecules (2, 62). Given that most, if not all, of these ssDNA replicons are evolutionarily related to and share many biological features with the geminiviruses (22, 31, 36), it is of interest to determine whether conserved recombination patterns observed in the geminiviruses (61, 81) are evident in these other groups. To date, no comparative analyses have ever been performed with different ssDNA virus families to identify, for example, possible influences of genome organization on recombination breakpoint distributions found in these viruses.Here we compare recombination frequencies and recombination breakpoint distributions in most currently described ssDNA viruses and satellite molecules and identify a number of sequence exchange patterns that are broadly conserved across this entire group.  相似文献   

20.
Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.  相似文献   

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