Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.In recent years, virus-like particles (VLPs), which are formed upon recombinant expression of the viral matrix and/or surface glycoproteins, have been recognized as representing powerful tools for developing novel vaccines and investigating certain aspects of the viral replication cycle (24, 44, 59, 63). Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16, 38, 42, 54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase. Expression of dominant-negative (DN) VPS4 mutants, which lack the ability to bind or hydrolyze ATP, blocks recycling of the ESCRTs and induces the formation of enlarged endosomes lacking internal vesicle accumulation (2, 3, 7). The inward budding of vesicles into the MVBs is topologically similar to the budding of viruses, since the vesicles bud away from the cytosol and into the lumen (reviewed in references 1, 20, and 26). Therefore, it is not entirely surprising that viruses use the cellular ESCRT machinery to organize the budding of viral progeny. Interactions between viral matrix proteins and ESCRTs occur through tetrapeptide motifs, known as late domains, which were first identified in retroviruses. Known late domains consist of the amino acid sequence P(T/S)AP, PPxY, or YxxL, where “x” represents any amino acid (19, 25, 62). The P(T/S)AP motif, for example, mediates interaction with tumor susceptibility gene 101 (Tsg101) (16, 36, 57); the PPxY motif mediates binding to WW domains of Nedd4-like ubiquitin ligases (9, 22); and the YxxL motif mediates interaction with AIP1/Alix (35, 47, 58). Recently, a novel late-domain motif, FPIV, has been identified in paramyxoviruses (46), and it is thought that additional late-domain motifs remain to be discovered (for a review, see reference 5).Inhibition of the VPS pathway has been shown to inhibit the budding of various viruses that are released with the help of ESCRTs. However, the budding of viruses and VLPs depends on the activity of ESCRTs to different degrees. Downregulation of Tsg101, a member of the ESCRT-I complex, inhibited the release of VLPs mediated by lymphocytic choriomeningitis virus Z protein and Marburg virus (MARV) VP40 (42, 54) but did not substantially inhibit the release of Gag-induced VLPs of Moloney murine leukemia virus and Rous sarcoma virus or that of matrix protein-induced VLPs of rabies virus (16, 27, 38). Expression of DN VPS4 inhibited the release of VLPs induced by the Gag proteins of Rous sarcoma virus and Moloney murine leukemia virus (16, 38) as well as that of VLPs induced by Lassa virus Z protein (55) but had no effect on the budding of rabies virus and cytomegalovirus (13, 27). These data indicate that in spite of the presence of late-domain motifs, a block in the VPS pathway may not always be critical for the budding of VLPs. In addition, the lack of known late domains in many enveloped viruses raises the question of whether they use other entry points into the VPS pathway or whether they exploit entirely different mechanisms of budding (60). To date, knowledge of how viral matrix proteins engage cellular machineries, such as the VPS pathway, to induce viral budding at the plasma membrane is very limited (8).The matrix protein VP40 of MARV contains only one known late-domain motif, PPPY, and a recent study showed that mutation of this late domain inhibited the release of VP40-induced VLPs. In addition, depletion of Tsg101 reduced the release of VP40-induced VLPs, suggesting that ESCRT-I is involved in this process (54). Whether a functional VPS pathway is important for the release of MARV VP40-induced VLPs or MARV particles remains unknown.VLPs induced by many viral matrix proteins have a morphology similar to that of cellular vesicles, which makes it difficult to separate the spherical VLPs from released cellular vesicles (4, 17, 53). In contrast, VLPs induced by the filovirus matrix protein VP40 are elongated and similar in morphology to viral particles (30, 49). Nevertheless, we observed that the supernatants of cells expressing VP40 contained various populations of particles with different morphologies. This raised the questions of whether the different particles are released by the same mechanism, whether they are all induced by VP40, and whether they are dependent on the same cellular pathways.The aim of the present study was to analyze the populations of particles released from cells expressing the MARV matrix protein VP40 and to gain further insights into the interaction between MARV and the cellular machinery involved in the budding of VLPs and MARV particles.We found that cells expressing VP40 released vesicular and filamentous particles, which could be separated by gradient centrifugation. Fractions with mainly vesicular particles represented a mixture of vesicles containing exclusively cellular proteins and vesicles also containing VP40 and few short filamentous particles. Longer filamentous particles, whose morphology resembled that of MARV particles but which displayed a much higher variability in length (400 nm to 5 μm), were found in denser gradient fractions. Filamentous VP40-induced VLPs were able to sort out cellular proteins efficiently. Release of VP40-induced filamentous VLPs was supported by the late-domain motif present in VP40, and inhibition of the cellular ESCRT machinery reduced the amount of these VLPs in the supernatant. Interestingly, the release of VLPs induced by a mutant of VP40 lacking the late domain was also reduced by inhibition of the cellular ESCRT machinery. Expression of a DN mutant of VPS4 diminished the budding of infectious MARV particles by 50%, a finding consistent with the idea that the activity of the ESCRT machinery supports viral budding but is not essential.     

Mumps Virus Matrix,Fusion, and Nucleocapsid Proteins Cooperate for Efficient Production of Virus-Like Particles

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.     

Intradermal Vaccination with Influenza Virus-Like Particles by Using Microneedles Induces Protection Superior to That with Intramuscular Immunization

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (≤40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.Influenza is a major health threat among infectious diseases, posing a significant burden for public health worldwide. Over 200,000 hospitalizations and approximately 36,000 deaths are estimated to occur annually in the United States alone (48, 49). Vaccination is the most cost-effective measure for controlling influenza. However, the influenza vaccine needs to be updated and manufactured every year due to changes in circulating viral strains. Current influenza vaccines rely on egg substrate-based production, a lengthy process with limited capacity that can cause shortages in available vaccine supplies. The recent 2009 outbreak of H1N1 influenza virus is a good example of the urgent need to develop a more effective vaccine platform and vaccination method (38).Influenza virus-like particles (VLPs) have been suggested as a promising alternative candidate to current influenza vaccines. Influenza VLPs are noninfectious particles that mimic the virus in structure and morphology, can be produced using an egg-free cell culture system, and have been shown to be highly immunogenic, inducing protective immunity (9, 15, 19, 27, 35, 41, 42, 44). Most current vaccines are administered intramuscularly to humans in liquid formulations using hypodermic needles or syringes. Another strategy to meet the potential need for mass vaccination would be to develop an effective method for vaccine delivery to the skin (4, 8, 32, 50, 52). The skin is considered an important peripheral immune organ rich in potent immune-inducing cells, including Langerhans cells (LCs), dermal dendritic cells (DCs), and keratinocytes (5, 13, 14, 22). LCs and DCs residing in the epidermal and dermal layers of the skin have been shown to play an important role in antigen processing and presentation following skin immunization (1, 13, 14, 22). Intradermal (ID) vaccination delivering antigens to the dermal layer of the skin has been performed in many clinical studies and have demonstrated dose-sparing effects in some cases (4, 28, 29). Particularly, ID delivery of vaccines might be more effective in the elderly population (50), the highest risk group for influenza epidemics (49). However, ID delivery of vaccines using hypodermic needles is painful and needs highly trained medical personnel. In addition, more frequent local reactions at the injection site were observed after ID delivery. Therefore, a simple and effective approach for vaccination without using hypodermic needles would be highly desirable.To overcome the skin barrier of the outer layer of stratum corneum, solid microneedles were previously coated with inactivated influenza viruses and used to successfully deliver vaccines to the skin, which provided protection comparable to that with conventional intramuscular immunizations (32, 52). Other vaccines have also been delivered using microneedles (17, 17a), but VLPs have never been used this way before. Delivery of a powdered form of inactivated influenza vaccines to the skin has also been demonstrated using a high-speed jet delivery device (10). These previous studies used high doses of vaccines, possibly due to the instability of vaccines in dry formulations.Influenza hemagglutinin (HA) is responsible for attachment of the virus to sialic acid-containing receptors on target cells. However, it is not well understood how functional activity of HA affects the immunogenicity of influenza VLP vaccines. For the first time in this study, we investigated the effect of HA stability, immune responses, and protective efficacies of solid-microneedle VLP vaccines containing H1 HA as a major influenza viral component after delivery to the skin in comparison to results with intramuscular immunization. We found that the functional integrity of HA in influenza VLPs significantly influenced the immunological and protective outcomes for both microneedle and intramuscular vaccination. In addition, we have observed differential outcomes contributing to the protective immunity by the delivery of HA-stabilized VLPs to the skin in terms of the types of immune responses, recall antibody responses, and viral clearance at an early time point after challenge compared to those induced by intramuscular immunization.     

Analysis of Modified Human Papillomavirus Type 16 L1 Capsomeres: the Ability To Assemble into Larger Particles Correlates with Higher Immunogenicity

L1 capsomeres purified from Escherichia coli represent an economic alternative to the recently launched virus-like particle (VLP)-based prophylactic vaccines against infection with human papillomavirus types 16 and 18 (HPV-16 and HPV-18), which are causative agents of cervical cancer. It was recently reported that capsomeres are much less immunogenic than VLPs. Numerous modifications of the L1 protein leading to the formation of capsomeres but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds or the deletion of helix 4. So far, the influence of these modifications on immunogenicity has not been thoroughly investigated. Here, we describe the purification of eight different HPV-16 L1 proteins as capsomeres from Escherichia coli. We compared them for yield, structure, and immunogenicity in mice. All L1 proteins formed almost identical pentameric structures yet differed strongly in their immunogenicity, especially regarding the humoral immune responses. Immunization of TLR4^−/− mice and DNA immunization by the same constructs confirmed that immunogenicity was independent of different degrees of contamination with copurifying immune-stimulatory molecules from E. coli. We hypothesize that immunogenicity correlates with the intrinsic ability of the capsomeres to assemble into larger particles, as only assembly-competent L1 proteins induced high antibody responses. One of the proteins (L1ΔN10) proved to be the most immunogenic, inducing antibody titers equivalent to those generated in response to VLPs. However, preassembly prior to injection did not increase immunogenicity. Our data suggest that certain L1 constructs can be used to produce highly immunogenic capsomeres in bacteria as economic alternatives to VLP-based formulations.Certain types of human papillomavirus (HPV) are the cause of cervical cancer, most frequently HPV types 16 and 18 (HPV-16 and HPV-18), which are responsible for about 50% and 20% of cases, respectively (8, 15, 16). Recently, two vaccines that prevent infection with HPV-16 and HPV-18 have been introduced to the market. These vaccines are based on the viral major structural protein L1, which can spontaneously self-assemble in vitro into empty virus-like particles (VLPs) that resemble the native virions in size and shape. VLPs have been shown to be highly immunogenic, as they can induce high titers of neutralizing antibodies (29, 30). HPV virions and VLPs consist of 72 L1 pentamers, also called capsomeres, which are arranged in an icosahedral T=7 particle lattice with a diameter of 55 nm. Cryo-electron microscopic analysis has revealed the presence of 60 hexavalent and 12 pentavalent capsomeres (4).Capsid assembly has been reported to be optimal at low pH (pH 5.4) and high ionic strength, whereas both high pH (pH 8.2) and the presence of reducing agents favor disassembly into capsomeres, the latter because the viral particles are stabilized by intercapsomeric disulfide bonds between two conserved cysteine residues at positions 175 and 428 (11, 35, 44). VLP formation is not affected by deletions of up to 9 amino acids (aa) from the N terminus and up to 34 aa from the C terminus of the L1 protein (11, 36). An N-terminally truncated L1 protein lacking 10 aa has been shown to assemble into particles consisting of 12 L1-pentamers with a T=1 lattice referred to as small VLPs (11, 12). Crystallographic analysis of the T=1 particles revealed that interpentameric contacts are established by hydrophobic interactions between the α-helices 2 and 3 of one capsomere and α-helix 4 of a neighboring capsomere (12). Consequently, a mutant L1 with helix 4 deleted formed homogenous capsomeres but failed in T=1 and T=7 particle assembly (7). Deletion of helices 2 and 3 impeded even pentamer formation, as a large fraction of the L1 protein was found to be insoluble, which suggests an essential role for these regions in L1 folding (7, 11).VLP-based prophylactic vaccines have been shown to induce high titers of neutralizing antibodies, which protect against virus challenge and associated diseases in humans (24, 31). However, due to the relatively high production and distribution costs of the vaccines—they are expressed in and purified from eukaryotic cells and require a cold chain for storage—they will probably be largely unavailable to developing countries, where more than 80% of all cervical cancer cases occur (1, 38, 46).L1 capsomeres represent a potentially lower cost alternative to VLPs, as they can be produced in large amounts from Escherichia coli and are considered more stable at room temperature (11, 34, 35). Capsomeres have been shown to induce high titers of neutralizing antibodies and T-cell responses upon oral, intranasal, and subcutaneous immunization and have also protected against viral challenge in the canine oral papillomavirus model (18, 19, 37, 42, 48, 53). Most of the immunization data for HPV capsomeres have been obtained from administration of full-length or N-terminally deleted (10 aa) wild-type L1 proteins (18, 37, 53). A recent report in which the L1 pentamers were derived from an L1 protein in which the conserved cysteines (aa 175 and 428) were replaced by alanines revealed that HPV-16 VLPs induce about 20- to 40-fold-higher humoral immune responses than capsomeres (47). The influence on immunogenicity of the other mutations and deletions of the L1 protein that prevent capsid assembly has so far not been studied in depth.In a comparative analysis of eight differently modified HPV-16 L1 proteins purified as capsomeres from E. coli, we now report that their potential to induce humoral immune responses in mice correlates with their ability to assemble into particles larger than capsomeres. One of the constructs, L1ΔN10, encoded for capsomeres that exhibited immunogenicity similar to that of VLPs.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

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

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

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Heterotypic Humoral and Cellular Immune Responses following Norwalk Virus Infection

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-γ) was measured. As seen with blockade responses, IFN-γ secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.Noroviruses are the second-most important cause of severe viral gastroenteritis in young children and cause approximately 20% of endemic familial diarrheal disease and traveler''s diarrhea in all ages (reviewed in references 45 and 70). Noroviruses are genetically grouped into five different genogroups (GI to GV). GI and GII genogroups are responsible for the majority of human infections and are subdivided into more than 25 different genotypes (for example, GI.1 is genogroup I genotype 1). Most norovirus outbreaks are caused by the GII.4 genotype (65). Although genogroup I strains are associated with fewer reported outbreaks, they are frequently identified in environmental samples and in children (7, 21, 33, 58, 74, 82). The severity of norovirus disease is usually moderate although infection can be especially virulent, even fatal, in the elderly (14, 24, 31, 38, 46, 67). An effective vaccine would be particularly advantageous to vulnerable older populations, food handlers, child and health care providers, and military personnel. One major obstacle to norovirus vaccine development is the lack of understanding of the extensive antigenic relationships between heterogenic norovirus family members and of how this antigenic heterogeneity affects host protective immunity. Norovirus heterogeneity can be examined through sequence, structural, ligand binding, and host immune studies.Structurally, noroviruses are ∼38-nm icosahedral viruses with an ∼7.5 kb single-stranded, positive-sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the replicase polyprotein, while ORFs 2 and 3 encode the major and minor capsid proteins, respectively. The ORF2 major capsid protein sequence can vary by up to 60% between genogroups and by ∼20 to 30% between the genotypes (91). Expression of the major capsid protein (ORF2) in baculovirus and Venezuelan equine encephalitis (VEE) results in formation of virus-like particles (VLPs) composed of 180 copies of the monomeric protein (72). The monomer is structurally divided into the shell domain (S) that forms the structural core of the particle and the protruding domain (P) that protrudes away from the core. The P domain is further subdivided into the P1 subdomain (residues 226 to 278 and 406 to 520) and the P2 subdomain (residues 279 to 405) (72). P2 represents the most exposed surface of the viral particle and determines interaction with both potential neutralizing antibody recognition sites and putative cellular receptors, the histo-blood group antigens (HBGAs) (13, 16, 54, 57).The P domain has been shown to independently form dimers and P particles comprised of 12 monomers (85). Dimers and P particles share structural and HBGA binding similarities with the VLP generated with the same monomers (9, 85, 87). Three norovirus-HBGA binding profiles have been identified: (i) those that bind A/B and/or H epitopes, (ii) those that bind Lewis and/or H epitopes, and (iii) those that do not bind any available HBGA (86). Elegant structural analyses of Norwalk virus VLPs in complex with synthetic HBGAs identified a highly conserved binding site within the G1 noroviruses and predicted that structural constraints within the GI strains would restrict HBGA binding patterns to either a terminal Gal-Fuc or GalNAc (18, 88).Norwalk virus (NV; GI.1-1968) is the prototypic GI strain and typically infects individuals who encode a functional FUT2 α-1,2-fucosyltransferase enzyme resulting in expression of HBGAs on mucosal surfaces (secretor-positive phenotype) (53). Individuals who do not encode a functional FUT2 enzyme have a secretor-negative phenotype, do not express ABH HBGAs on mucosal surfaces, and are resistant to NV infection. Outbreak investigations have confirmed the association between HBGA expression and norovirus infection for some GI and GII strains (37, 39, 43, 49, 89). It remains likely that enzymes other than FUT2 may function as norovirus susceptibility factors because secretor-negative individuals have low-level norovirus-reactive antibodies (49, 52, 53) and can become infected after challenge with a GII.2 strain (52); in addition, some norovirus strains bind to FUT2-independent HBGAs in vitro (35, 54, 79).Early challenge studies (reviewed in reference 50) suggested that short-term protective immunity may occur following NV challenge (96). Demonstration of long-term protective immunity has been more complex. One early rechallenge study found that 50% of NV-challenged volunteers experienced repeat infections after ∼3 years while the other 50% remained well initially and after repeated challenge (69). Whether these volunteers remained disease free because of acquired immunity or genetic resistance could not be ascertained (69). However, contemporary norovirus challenge studies suggest that an early mucosal IgA response is associated with protection from NV infection (53). Further, strong gamma interferon (IFN-γ) secretion from CD4⁺ T cells (52) was identified in some uninfected GII.2-1976-challenged volunteers.In the absence of additional rechallenge studies, the most compelling evidence for a long-term protective immune response comes from the growing number of reports from around the world indicating that periods of “high norovirus activity” correlated with the emergence of new GII.4 strains (1, 10, 42, 66, 75, 90). Subsequently, the years following the high activity were characterized by decreased numbers of outbreaks, indicating that herd immunity may be an important regulator of GII.4 noroviruses (54, 80, 81). Clearly, the molecular basis for differential protective immunity/susceptibility following repeat norovirus infection is complex and a major challenge for the field.In this report, we compare the VLP phenotypes of the prototypical norovirus strain NV to an extant GI.1 strain isolated 33 years after NV and to a panel of VLPs representing strains GI.2, GI.3, and GI.4. In the results, we evaluate sequence conservation, carbohydrate (CHO) binding patterns, and antigenic relatedness at the antibody and T-cell levels. In contrast to earlier predictions (19), these data suggest that the GI noroviruses can bind many different HBGAs and that individuals infected with norovirus usually mount robust B- and T-cell responses against homologous strains. Surprisingly, some individuals appear to preferentially mount immune responses against heterologous GI strains.     

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.Since the first detection of the Asian lineage of highly pathogenic avian influenza (HPAI) virus (H5N1) in southern China in 1996, H5N1 virus infection in birds has continued for 13 years in Asia, acquiring pathogenicity not only in birds but also in mammals. In 1997, the H5N1 Hong Kong isolates caused illness and death in a variety of terrestrial birds and even in humans (9, 37, 48, 49). In 2001, emerging H5N1 Hong Kong isolates were more pathogenic to chickens and the mean death time (MDT) was 2 days without any prior clinical signs (12). In 2003 to 2004, the H5N1 epizootic occurred simultaneously in East Asian countries (22, 30). The 2003/2004 H5N1 isolates caused death in taxonomically diverse avian species, including domestic ducks (46, 47, 51), and humans (7, 55). Furthermore, the first indication of wild aquatic bird involvement occurred at recreational parks in Hong Kong in late 2002 to 2003 (46), and then migratory aquatic bird die-off occurred in 2005 at Qinghai Lake in China (6, 24). The broad host spectrum and increased pathogenicity of H5N1 viruses to diverse bird species raise serious concerns about the worldwide spread of the virus by migratory birds.According to the international criteria, HPAI viruses are defined by over 75% mortality in 4- to 8-week-old chickens following an intravenous pathogenicity test or an intravenous pathogenicity index (IVPI) of more than 1.2 in 6-week-old chickens (34); however, there are some variations in pathogenicity intensity among the HPAI viruses in chickens (1, 3, 5, 12, 15, 28, 31, 48, 50-52, 57). Most of the HPAI viruses that were isolated before 1996 cause severe clinical signs (e.g., ruffled feathers, depression, labored breathing, and neurological signs) and severe gross lesions (e.g., head and face edema, cyanosis, subcutaneous hemorrhages in combs and leg shanks, and necrosis of combs and wattles) in chickens (1, 3, 15, 31, 50, 52, 57). These viruses usually kill chickens 3 to 6 days after intranasal inoculation. On the other hand, the recently emerged Asian H5N1 HPAI viruses are more virulent and kill chickens within 1 to 2 days without causing typical clinical signs and gross lesions (5, 12, 27, 33, 48, 51), although some Asian H5N1 viruses, such as A/Goose/Guangdong/2/96 (23), A/goose/Hong Kong/437-10/99 (17), and A/chicken/Indonesia/7/03 (58), were less virulent. To successfully control HPAI in poultry, it is important to better understand the mechanisms of increased pathogenicity of recent H5N1 HPAI viruses in chickens.The Asian H5N1 HPAI virus has another important characteristic, which is its capability of crossing host-species barriers. It was reported that the H5N1 virus can infect and cause death in mammals such as mice (5, 9, 12, 14, 29), cats (21), tigers (2), ferrets (11, 26), monkeys (40), and humans (7, 49, 55). High-level inductions of proinflammatory cytokines in mammals infected with the H5N1 viruses, referred to as “cytokine storms,” have been hypothesized to contribute to the severity of pathological changes and ultimate death (4, 7, 13, 45, 55). Cytokine and chemokine dysregulation was detected in clinical cases of H5N1-infected humans (8, 13, 36) and also in monkeys experimentally infected with the H1N1 Spanish flu strain (20). In a mouse model, lymphocyte apoptosis and cytokine dysregulation have been proposed to contribute to the severity of the disease caused by H5N1 (56). Investigations with transgenic mice deficient in each cytokine gene suggest that tumor necrosis factor alpha (TNF-α) may contribute to morbidity and interleukin-1 (IL-1) may be important for virus clearance (53). However, mice deficient in TNF-α or IL-6 succumb to infection with H5N1, and cytokine inhibition treatment does not prevent death (42), suggesting that therapies targeting the virus rather than cytokines may be preferable. Thus, the significance of elevated proinflammatory cytokine responses in the pathogenesis of H5N1-infected mammals requires further studies.In contrast, little is known about proinflammatory cytokine responses and their roles in pathogenicity in chickens infected with HPAI viruses, including the recent Asian H5N1 viruses. It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-γ) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown. The less-virulent Asian H5N1 virus, which causes severe clinical signs and gross lesions in chickens (17, 23, 27, 58), would be a valuable tool for investigating the role of proinflammatory cytokines in chickens infected with HPAI viruses, as well as for exploring the pathogenesis of the more-virulent Asian H5N1 HPAI virus, because of the antigenic and molecular similarities between them.In this study, we compared the pathogenicities in chickens of the less-virulent and more-virulent Asian H5N1 HPAI viruses based on MDT, fever, cytokine responses, and viral replication. Our results suggest that the shift in the Asian H5N1 virus to increased virulence may be associated with efficient and rapid replication of the virus in chickens, accompanied by early destruction of host immune responses and followed by peracute death before fever can be induced. Finally, we discuss candidate genes that may account for the high pathogenicity of Asian H5N1 HPAI viruses in chickens.