MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.West Nile virus (WNV) is an arthropod-borne virus that is a member of the Flaviviridae family, which includes St. Louis encephalitis virus, Kunjin virus, yellow fever virus, dengue virus, and Murray Valley encephalitis virus (2). Since its first identification in the West Nile province of Uganda in 1937, WNV has spread quickly through Asia, Europe, and the United States and has caused a serious global health problem (34). The clinical manifestations of WNV usually entail neurological diseases such as meningitis and encephalitis. This might be caused by WNV genome replication after inoculation and its subsequent spread to lymph nodes and blood, followed by its entrance into the central nervous system through Toll-like receptor and tumor necrosis factor receptor (40).WNV has the genome of a single positive-sense RNA containing one open reading frame. The encoded polypeptide is processed further by viral and cellular proteases into several nonstructural and structural proteins (2). Nonstructural (NS) proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS1 is involved in synthesis of viral RNA, and NS3 mediates the cleavage of nonstructural proteins (22, 25, 30, 48). NS5 functions as an RNA polymerase and methyltransferase, which are required for viral replication (14, 17, 18). NS2A, NS2B, NS4A, and NS4B promote the organization of viral replication factors and membrane permeabilization (3, 5, 6, 13, 37). The capsid, envelope (E), and premembrane (prM) proteins are the structural proteins, which are involved in virus assembly (43). E protein is a virion surface protein that regulates binding and fusion to the cell membrane (1, 11, 32). The prM protein is a precursor of the M protein, which is translocated to the endoplasmic reticulum (ER) by capsid (2, 21). Viral assembly occurs mainly in the ER membrane following release of viral particles (23).The capsid of WNV (WNVCp) localizes and is involved in nucleocapsid assembly on the ER membrane (15). However, extra roles of the flavivirus capsid in the nucleus has been reported. For example, capsid proteins of Japanese encephalitis virus (JEV) and hepatitis C virus (HCV), which are also members of the Flaviviridae family, participate in pathogenesis by localizing to the nucleus (33). Nucleolar and nuclear WNVCp is involved in pathogenesis via induction of the apoptotic process in cells through interaction with Hdm2, which results in the activation of the potent tumor suppressor p53 (47). It also induces apoptotic death of neuron cells via mitochondrial dysfunction and activation of caspase pathways when introduced into the brains of mice (46).The Makorin ring finger protein 1 (MKRN1) gene was first reported as the source gene of introns for the intronless imprinted MKRN gene family (10). The protein is an ancient protein conserved from invertebrates to vertebrates, and it contains several zinc finger motifs, including C3H, C3HC4, and unique Cys-His motifs (10). Furthermore, this gene is constitutively expressed in most human tissues, including neurons (10). The role of MKRN1 as an E3 ligase was first identified by its ability to degrade hTERT (16). Interestingly, MKRN1 functions as a coregulator of androgen and retinoic acid receptor (27), suggesting possible diverse roles of MKRN1 in human cells.In this study, we report on an ubiquitin (Ub) E3-ligase for WNVCp. MKRN1 was able to ubiquitinate and degrade WNVCp in a proteasome-dependent manner. Furthermore, degradation of WNVCp resulted in a reduction of WNV-induced cell death. Cells stably overexpressing MKRN1 were resistant to WNV-induced cell death. In contrast, ablation of MKRN1 by small interfering RNA (siRNA) renders cells more susceptible to the cytotoxicity of WNVCp. Furthermore, WNV proliferation was suppressed in 293T cells overexpressing MKRN1 but increased in MKRN1-depleted 293T cells. Based on these data, we suggest that MKRN1 might play a role in protection of cells against WNV infection.     

Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.West Nile encephalitis virus (WNV) is a positive-polarity, single-stranded RNA virus of the genus Flavivirus within the family Flaviviridae. Other members of this genus that cause significant human disease include dengue virus (DENV), St. Louis encephalitis virus, Japanese encephalitis virus (JEV), yellow fever virus, and tick-borne encephalitis virus (TBEV). Flaviviruses are translated as a single polypeptide, which is then cleaved by host and viral proteases into three structural (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (reviewed in references 42 and 43).WNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as dead-end hosts (25, 62). Infection causes syndromes ranging from a mild febrile illness to severe encephalitis and death (13, 72). WNV has spread globally and causes outbreaks with thousands of severe human cases annually in the United States. An age of greater than 55 years, a compromised immune status, and a CC5Δ32 genotype have been associated with more-severe disease (15, 20). There is currently no approved vaccine or therapy for WNV infection.The mature WNV virion has a ∼500-Å diameter and consists of a single RNA genome surrounded by the capsid protein, a lipid bilayer, and a shell of the prM/M and E proteins (31, 55). X-ray crystallography studies have elucidated the three-domain structure of the flavivirus E protein (30, 48, 50, 58, 67). Domain I (DI) is a central, eight-stranded β-barrel, which contains the only N-linked glycosylation site in WNV E. Domain II (DII) is a long, finger-like protrusion from DI and contains the highly conserved fusion peptide at its distal end. Domain III (DIII) adopts an immunoglobulin-like fold at the opposite end of DI and is believed to contain a site for receptor attachment (6, 8, 40).Within an infected cell, progeny WNV are assembled initially as immature particles. In immature virions, three pairs of E and prM interact as trimers and form 60 spiked projections with icosahedral symmetry (85, 86). Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of the E proteins and cleavage of prM to M by a furin-like protease (41, 83). Mature WNV virions are covered by 90 antiparallel E protein homodimers, which are arranged flat along the surface in a herringbone pattern with quasi-icosahedral symmetry (55).Upon binding to poorly characterized cell surface receptors, internalization of WNV is believed to occur through receptor-mediated, clathrin-dependent endocytosis (1, 79, 80). After trafficking to Rab5- and/or Rab7-positive endosomes (38, 79), the mildly acidic pH within the lumen of the endosome induces structural alterations in the flavivirus E protein (7, 49), which includes changes in its oligomeric state (7, 49, 77). During this process, also known as type II fusion, the hydrophobic peptide on the fusion loop of DII of the E protein inserts into the endosomal membrane, thus physically joining the host and viral membranes, which allows the infectious RNA genome to enter the cytoplasm (32, 33).Humoral immunity is an essential component of the protective host response against flaviviruses including WNV (reviewed in references 64 and 68). Studies by several groups have shown that the neutralization of WNV can occur after antibodies bind to a series of discrete epitopes on all three domains of the E protein (3, 12, 22, 59, 61, 71). To date, the most potently neutralizing monoclonal antibodies (MAbs) localize to an epitope on the lateral ridge of DIII (DIII-lr). One well-characterized strongly neutralizing mouse MAb, E16, blocks infection primarily at a postattachment step (57) and requires the engagement of only a fraction of its epitopes on the surface of the virion (66). Studies of the human antibody response to WNV infection reveal that, in contrast to mice, antibodies that bind the DIII-lr epitope comprise a minor component of the neutralizing humoral response in most individuals (60).In this study, we characterized two strongly neutralizing novel human MAbs (CR4348 and CR4354) that were selected from an antibody phage display library constructed from B cells of subjects that survived WNV infection (78). We demonstrate that both MAbs are WNV specific, bind weakly to recombinant or yeast surface-displayed E proteins, exhibit pH-sensitive binding to viral particles, and protect against lethal infection in mice. Our experiments suggest that these human MAbs map to distinct epitopes and neutralize infection at a postattachment stage, likely by inhibiting the acid-catalyzed viral fusion step.     

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause invasive neurological diseases and lethal hemorrhagic fever in humans, respectively (6, 32). Since its first incursion into New York City in 1999, WNV has rapidly spread throughout the continental United States and has recently reached South America (29, 34). In most cases, WNV infection of people resolves as an asymptomatic or a mild febrile illness. However, approximately 1% of infections result in severe neurological disorders, such as encephalitis and meningitis (27). Unlike WNV, for which people are only accidental hosts, DENV has fully adapted to humans (32). It has apparently lost the need for an enzootic cycle and causes a range of diseases in people, from acute febrile illness to life-threatening dengue hemorrhagic fever/dengue shock syndrome (6). Four distinct serotypes of DENV have spread throughout the tropical and subtropical parts of the world, with an estimated 50 to 100 million human cases annually and about 2.5 billion people worldwide being at risk of infection (32). Effective antiviral therapies and vaccines to treat or prevent WNV and DENV infections in humans are not yet available.Type I interferons (IFNs), represented by IFN-α and IFN-β, have been demonstrated to play an essential role in defending against WNV and DENV infections. For example, mice with deficiencies in the induction of type I IFNs and the receptor or JAK-STAT signal transduction pathway of the cytokines are vulnerable to WNV and DENV infections (7, 38, 42, 49-51). In addition, a strain of WNV that fails to block the type I IFN signal transduction pathway is phenotypically attenuated in mice (23, 50). Clinically, during acute DENV infection, innate immune responses play a key role in determining disease outcome (35), and resolution of WNV infection requires effective IFN-mediated innate host responses (23, 43, 53). Therefore, understanding how the IFN-mediated innate immune response functions is one of the critical frontiers in the molecular biology of WNV and DENV pathogenesis (1, 44).IFNs inhibit virus infection by induction of IFN-stimulated genes (ISGs) that disrupt distinct steps of the viral replication cycle (47). However, although IFN treatment of cells induces the expression of hundreds of cellular genes (9), only approximately a dozen ISGs have been experimentally demonstrated to instigate an antiviral state against selected viruses (41). As mentioned above, although there is ample evidence suggesting that IFN-mediated innate immunity plays a critical role in defending against WNV and DENV infections, the underlying antiviral mechanism of the cytokines remains to be understood (6, 16, 31). With WNV, previous studies suggested that mice lacking double-stranded-RNA-activated protein kinase (PKR) and RNase L were more susceptible to the virus infection and had increased viral loads in multiple peripheral organs and neuronal tissues, in comparison with congenic wild-type mice (43). In addition, genetic studies showed that a nonsense mutation in the gene encoding the 2′,5′-oligoadenylate synthetase 1b (OAS1b) isoform was associated with WNV susceptibility in mice, and expression of wild-type OAS1b in mouse fibroblasts efficiently inhibited WNV infection (22, 33, 37, 45). For DENV, it was reported recently that viperin was among the highly induced ISGs in DENV-infected cells and overexpression of viperin in A549 cells significantly reduced DENV replication (13).In principle, to understand how IFNs inhibit DENV and WNV infections, it is essential to know the repertoire of ISGs that are directly implicated in antiviral action and understand how these antiviral ISGs work individually and coordinately to limit virus replication. To achieve this goal, we set out to systematically identify the ISGs that are able to inhibit infection with the two viruses and elucidate their antiviral mechanisms.     

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.The genus Flavivirus, many of whose more than 70 members are arthropod-borne human pathogens, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus, and Japanese encephalitis virus (JEV), has assumed increasing public health importance in recent years. The single-strand, positive-sense RNA genomes of flaviviruses encode a single polyprotein, which is cotranslationally cleaved to produce three structural proteins (capsid [C], membrane [M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). NS1, a glycoprotein, is perhaps the most versatile among these, being involved both in vital processes such as viral RNA synthesis and in multiple interactions with the host, in ways that appear to benefit both pathogen and host. Following translocation into the lumen of the endoplasmic reticulum through a hydrophobic signal sequence that is encoded by the carboxyl terminus of E (17), NS1 undergoes glycosylation followed by rapid dimerization (44, 52). In DENV infection of cultured mammalian cells, extracellular NS1 was additionally detected as hexamers (19, 51). Despite the apparent absence of a canonical hydrophobic membrane anchor domain, the NS1s from JEV, Kunjin virus, DENV, and YFV have all been shown to be present on the surface of virus-infected cells (8, 23, 50). The mechanistic details of this membrane anchor still remain uncertain.The ability of DENV NS1 to bind host complement (9, 49) pointed to a role for this protein in DENV pathogenesis. Serum NS1 levels in both DENV and WNV patients correlate directly with disease severity (1, 36). Promotion of immune complex formation (54), ability to elicit autoantibodies with reactivity to platelets and extracellular matrix (10), and damage inflicted on endothelial cells (34) are some of the mechanisms proposed to explain pathogenesis mediated by DENV NS1. Recent studies with WNV NS1 demonstrated its ability to bind human complement factor H, suggesting a role in reducing the host''s ability to bring about complement-mediated control of early virus replication (11).Critical differences between the functions of NS1s encoded by different pathogenic flaviviruses and their contributions to pathology are evident from the published reports, with DENV NS1 believed to be involved in complement activation and the consequent capillary leak syndrome of dengue hemorrhagic fever (6), while WNV NS1 appears relatively more benign and has more to do with modulation of the host innate immune response (11). We have not encountered reports of adverse impacts of JEV NS1 in infected individuals.Paradoxically, several studies have pointed to a role for flavivirus NS1-specific immune responses in protection against flaviviruses. Passive immunization studies using monoclonal antibodies (MAbs) (24, 28, 29, 55) as well as immunization of mice using naked DNA constructs expressing NS1 (35, 40) revealed that antibodies directed to prM or E of DENV and NS1 of DENV and JEV are protective. Studies by different groups have shown that active immunization with purified NS1 or passive immunization with MAbs against YFV and DENV NS1 provides protection from lethal viral challenge in the absence of neutralizing antibodies (24, 45, 48). A panel of anti-WNV NS1 MAbs revealed multiple antibody-mediated mechanisms for protection, some mediated through complement and others via the Fc receptor (12). Those authors went on to show that anti-NS1 MAbs that facilitate phagocytosis and clearance of WNV-infected cells through Fc-γ receptors I and/or IV belonged to the IgG2a subclass and bound to cell surface-associated NS1 (13).Earlier studies also pointed to the cytolytic potential of NS1 antibodies, a property that might contribute significantly to their protective ability. Passive immunization experiments using a panel of anti YFV NS1-specific MAbs showed a significant correlation between protection and in vitro complement-mediated cytolysis of YFV-infected mouse neuroblastoma cells (47). Additionally, immunization of mice with a DNA vaccine construct carrying JEV NS1 induced a strong antibody response exhibiting complement-mediated cytolysis of JEV-infected cells (35), but no neutralizing activity, and resulted in protection against subsequent challenge with virus. Cell-mediated immune responses directed to NS1 of JEV have also been reported to play a role in cytotoxic T-lymphocyte-mediated killing of JEV-infected murine target cells (41). Thus, NS1 appears to contribute to protection in the murine model by inducing both humoral and cell-mediated arms of the immune response.It was therefore of interest to query whether NS1-specific antibodies in humans exposed to JEV exhibit cytolytic activity and to determine if these antibodies are capable of reducing virus production by infected cells. In this study we report for the first time the existence of detectable levels of anti-NS1 antibodies in a significant proportion of sera from humans infected with JEV and demonstrate their ability to induce antibody-dependent complement-mediated cytolysis of cells expressing JEV NS1 on the surface. These sera failed to cause lysis of cells infected with WNV or DENV, both of which cocirculate with JEV in the Indian subcontinent and have been reported in the region where we enrolled our volunteers, revealing stringent specificity and absence of flaviviral cross-reactivity for these cytolytic antibodies. Furthermore, we demonstrate the ability of NS1-specific antibodies elicited in mice to limit virus production in infected human SW-13 cell monolayers, which may explain, at least in part, the widely reported protective ability of flavivirus NS1. Significantly, we found no evidence for the ability of NS1 from JEV to bind human complement factor H, in contrast to the case for WNV NS1 (11). Taken together, these findings suggest that JEV NS1 may positively and significantly affect virus-specific protective immune responses.     

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.Recent estimates predict that the prevalence of hepatitis C virus (HCV) infection is approximately 2.2% worldwide, equivalent to about 130 million persons (22). The virus typically establishes a chronic infection that frequently leads to serious liver disease (1), and current models indicate that both morbidity and mortality as a consequence of HCV infection will continue to rise for about the next 20 years (10, 11, 29).HCV is the only assigned species of the Hepacivirus genus within the family Flaviviridae. The virus can be classified into six genetic groups or clades (numbered 1 to 6) and then further separated into subtypes (e.g., 1a, 1b, 2a, 2b, etc.) (53, 55). HCV has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb in length (reviewed in reference 46). Genomic RNA carries a single open reading frame flanked by 5′ and 3′ nontranslated regions, which are important for both replication and translation (19, 20, 34, 47, 56). Viral RNA is translated by the host ribosomal machinery, and the resultant polyprotein is co- and posttranslationally cleaved to generate the mature viral proteins. The structural proteins (core, E1, and E2) and a small hydrophobic polypeptide called p7 are produced by the cellular proteases signal peptidase and signal peptide peptidase (28, 45, 54). Two virus-encoded proteases, the NS2-3 autoprotease and the NS3 serine protease (5, 13, 26), are responsible for maturation of the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). With the exception of NS2, the NS proteins are necessary for genome replication (8, 40) and form replication complexes (RCs), which are located at the endoplasmic reticulum (ER) membrane (14, 24, 52, 57, 59). The functions of all viral constituents of RCs have not been characterized in detail. It is known that NS5B is the RNA-dependent RNA polymerase (6), while NS3 possesses helicase and nucleoside triphosphatase activities in addition to acting as a protease (32, 58). However, the precise roles of the other proteins remain to be firmly established.Expression of NS4B, one of the replicase proteins, generates rearrangements at the ER membrane that have been termed the “membranous web” (14, 24) and “membrane-associated foci” (MAFs) (25). Detection of viral RNA at such foci suggests that NS4B is involved in creating the sites where genome synthesis occurs (18, 24, 59). It is predicted that NS4B has an amphipathic α-helix within its N-terminal region, which is followed by four transmembrane domains (TMDs) in the central portion of the protein (17, 42). As a result, the majority of NS4B is likely to be tightly anchored to membranes, and experimental evidence indicates that it has characteristics consistent with an integral membrane protein (27). It is thought that after membrane association, NS4B rearranges membranes into a network, thereby generating foci which act as a “scaffold” to facilitate RNA replication. The mechanisms engaged in formation of foci are not known but include the notion that the NS4B N terminus can translocate into the ER lumen, resulting in rearrangement of cellular membranes (41, 42). Alternatively, palmitoylation, a lipid modification, might facilitate polymerization of NS4B, in turn promoting formation of RCs on the ER membrane (68).Apart from inducing membranous changes required for replication, NS4B may perform other tasks in HCV RNA synthesis. For example, studies of cell culture adaptive mutations in subgenomic replicons (SGRs) have identified amino acid changes that can stimulate RNA production (39), suggesting that NS4B may exert a regulatory role in determining replication efficiency. In support of a regulatory function, replacement of NS4B sequences in an SGR from strain H77 (a genotype 1a strain) with those from strain Con-1 (a genotype 1b strain) gave higher levels of replication than for a wild-type (wt) strain H77 SGR (7). The corresponding replacement of strain Con-1 NS4B sequences with those from strain H77 reduced the replication efficiency of a Con-1 SGR (7). Moreover, interactions of NS4B with the RC can affect the behavior of other replicase proteins. For example, NS4B is needed for hyperphosphorylation of NS5A (35, 48) and restricts its intracellular movement (30).To try to gain greater insight into the functional organization of the components that constitute RCs, trans-complementation assays using defective and helper SGRs have been established (2, 64). Such studies reveal that the only protein capable of trans-complementation is NS5A, while active replication cannot be restored for replicons harboring deleterious mutations in NS3, NS4B, and NS5B. These data led to the conclusion that functional NS5A may be able to exchange between RCs (2), whereas, by inference, such exchange would not be possible for other HCV replicase proteins. In transient-replication assays, complementation by NS5A also relied on its expression as part of a polyprotein (minimally NS3-NS5A), and production of the protein alone failed to restore replication for an inactive SGR (2). However, in a separate study, stable expression of wt NS5A was capable of complementing a defective replicon (64). Thus, different assay systems can give dissimilar results for complementation by NS5A.In this study, we have created a series of mutations in the NS4B gene of HCV strain JFH1 (31) to explore the function of the protein in the HCV life cycle. We focused our attention on the C-terminal portion of NS4B, downstream from the predicted TMD regions, since it is relatively well conserved and is predicted to lie on the cytosolic side of the ER membrane (15, 42). Our analysis examines the impact of mutations on replication efficiency and the intracellular characteristics of the mutants compared to the behavior of the wt protein. In addition, we have utilized this series of mutants to reassess trans-complementation of NS4B in replication assays. Finally, we also analyze the impact of mutations which do not affect replication on the production of infectious virus to determine whether NS4B plays a role in virus assembly and release.     

Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine''s anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA''s PPIase activity in the proper assembly and function of the HCV RC.Hepatitis C virus (HCV), of the family Flaviviridae, is an enveloped, positive-stranded RNA virus. Spread mostly by blood-borne transmission, HCV infects more than 170 million people worldwide. The viral genome is composed of a single open reading frame (ORF) plus 5′- and 3′-nontranslated regions. The ORF encodes a large polyprotein that is cleaved by cellular and viral proteases into 10 viral proteins. The structural proteins, including the capsid protein (core), two glycoproteins (E1 and E2), and a small ion channel protein (p7), reside in the N-terminal half of the polyprotein. The rest of the ORF encodes six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3 through NS5B assemble into a replication complex (RC) and are necessary and sufficient for HCV RNA replication in cell culture (8, 42). NS3 is a multifunctional protein with both a serine protease and an RNA helicase activity. The protease activity is responsible for cleavage at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (5), and the helicase activity is probably required to unwind the double-stranded RNA intermediates formed during replication (38). NS4A serves as an essential cofactor for the NS3 protease and anchors the NS3 protein to intracellular membranes (25, 36, 39). NS4B induces the formation of a “membranous web” that is probably the site of HCV replication (16). It also contains a GTP-binding motif that is required for replication (17). The web is derived from the endoplasmic reticulum (ER) compartment, although proteins of early-endosome origin have also been found to locate to the web (62). NS5A is a phosphoprotein and an integral component of the viral RC. The precise function of NS5A in replication is still unknown but appears to be regulated by phosphorylation and its interaction with several cellular proteins (19, 22, 24, 51, 52, 59, 63, 67). In addition, it may be involved in the transition from replication and particle formation (4, 45, 64). NS5B is the RNA-dependent RNA polymerase that is responsible for copying the RNA genome of the virus during replication. Several cellular cofactors interact with NS5B and modulate its activity in the context of the viral RC (22, 24, 35, 69, 71).Positive-stranded RNA viruses alter the intracellular membranes of host cells to form an RC in which RNA replication occurs. Modifications include the proliferation and reorganization of certain cellular membranes (1). HCV forms an RC associated with altered cellular membranes (16, 23), and crude RCs (CRCs) that maintain the replicase activity in vitro can be isolated by membrane sedimentation or flotation techniques (2, 3, 18, 27, 37).Cyclosporine is a widely used immunosuppressive and anti-inflammatory drug for organ transplant patients. It functions by forming an inhibitory complex with cyclophilins (CyPs) that inhibits the phosphatase activity of calcineurin, which is important for T-cell activation. In recent years, cyclosporine and its derivatives have been shown to be highly effective in suppressing HCV replication in vitro (44, 49, 53, 68) and in vivo (30). The mechanism of this inhibition is independent of its immunosuppressive function and distinct from that of interferon (IFN) (44, 53, 56, 68).We recently showed that HCV infection in vitro is inhibited when CyPA, a major intracellular target of cyclosporine, is downregulated by RNA interference, and mutations in NS5B that confer cyclosporine-resistant binding to CyPA contribute to the cyclosporine resistance of the replicons harboring these mutations (56, 71). Here we report that CyPA is recruited into the HCV RC together with NS5B in HCV replicon or in HCV-infected cells. Cyclosporine disrupts the association between RC-incorporated NS5B and CyPA and results in an exclusion of the polymerase from the viral RC. We also show that the prolyl-peptidyl isomerase (PPIase) motif of CyPA is essential for HCV replication.     

A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) are causative agents of economically important livestock diseases. Together with the genera Flavivirus, including several important human pathogens like Dengue fever virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus, and Hepacivirus (human Hepatitis C virus [HCV]), the genus Pestivirus constitutes the family Flaviviridae (8, 20). All members of this family are enveloped viruses with a single-stranded positive-sense RNA genome encompassing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTR) (see references 8 and 28 for reviews). The ORF encodes a polyprotein which is co- and posttranslationally processed into the mature viral proteins by viral and cellular proteases. For BVDV, the RNA genome is about 12.3 kb in length and encodes a polyprotein of about 3,900 amino acids. The first third of the ORF encodes a nonstructural (NS) autoprotease and four structural proteins, while the remaining part of the genome encodes NS proteins which share many common characteristics and functions with the corresponding NS proteins encoded by the HCV genome (8, 28). NS2 of BVDV represents a cysteine autoprotease which is distantly related to the HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and NS5B are essential components of the pestivirus replicase (7, 10, 49). NS3 possesses multiple enzymatic activities, namely serine protease (48, 52, 53), NTPase (46), and helicase activity (51). NS4A acts as an essential cofactor for the NS3 proteinase. NS5B represents the RNA-dependent RNA polymerase (RdRp) (22, 56). The functions of NS4B and NS5A remain to be determined. NS5A has been shown to be a phosphorylated protein that is associated with cellular serine/threonine kinases (44).According to their effects in tissue culture, two biotypes of pestiviruses are distinguished: cytopathogenic (cp) and noncytopathogenic (ncp) viruses (17, 27). The occurrence of cp BVDV in cattle persistently infected with ncp BVDV is directly linked to the induction of lethal mucosal disease in cattle (12, 13). Previous studies have shown that cp BVDV strains evolved from ncp BVDV strains by different kinds of mutations. These include RNA recombination with various cellular mRNAs, resulting in insertions of cellular protein-coding sequences into the viral genome, as well as insertions, duplications, and deletions of viral sequences, and point mutations (1, 2, 9, 24, 33, 36, 37, 42). A common consequence of all these genetic changes in cp BVDV genomes is the efficient production of NS3 at early and late phases of infection. In contrast, NS3 cannot be detected in cells at late time points after infection with ncp BVDV. An additional major difference is that the cp viruses produce amounts of viral RNA significantly larger than those of their ncp counterparts (7, 32, 50). While there is clear evidence that cell death induced by cp BVDV is mediated by apoptosis, the molecular mechanisms involved in pestiviral cytopathogenicity are poorly understood. In particular, the role of NS3 in triggering apoptosis remains unclear. It has been hypothesized that the NS3 serine proteinase might be involved in activation of the apoptotic proteolytic cascade (21, 55). Furthermore, it has been suggested that the NS3-mediated, enhanced viral RNA synthesis of cp BVDV and subsequently larger amounts of viral double-stranded RNAs may play a crucial role in triggering apoptosis (31, 54).In this study, we describe generation and characterization of a temperature-sensitive (ts) cp BVDV mutant whose ability to cause viral cytopathogenicity at high temperature is strongly attenuated. Our results demonstrate that a single amino acid substitution in NS2 attenuates BVDV cytopathogenicity at high temperature without affecting production of infectious viruses and expression of NS3 in a temperature-dependent manner.     

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31, 46, 57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5′ type 1 cap and conserved RNA structures at both the 5′ and 3′ untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. ​(Fig.1).1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, NS3, and NS5 are cytoplasmic. NS2A, NS2B, NS4A, and NS4B are largely hydrophobic transmembrane proteins with small hydrophilic segments (Fig. ​(Fig.1).1). The post- and cotranslational cleavage of the polyprotein is performed by NS3 in the cytoplasm and by host proteases in the ER lumen to yield the mature proteins (Fig. ​(Fig.1)1) (33, 43). Of the nonstructural proteins, NS3 and NS5 are the best characterized, and both are essential for viral replication (23, 27, 41). Both proteins are multifunctional. The N-terminal one-third of NS3 contains the viral protease (NS3pro), which requires a portion of NS2B for its activity, while the remaining portion codes for the RNA helicase/NTPase/RTPase domain (NS3hel) (21, 22, 32, 55). NS5, however, contains both an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (16, 51). The functions of NS1, NS2A, NS4A, and NS4B are not well understood, but they appear to play important roles in replication and virus assembly/maturation and have been found to bind to NS3 and NS5, possibly modulating their activity (33, 36).Open in a separate windowFIG. 1.Schematic diagram of flavivirus polyprotein organization and processing. (Top) Linear organization of the structural and nonstructural proteins within the polyprotein. (Middle) Putative membrane topology of the polyprotein predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (indicated by arrows). (Bottom) Different complexes that are thought to arise in different cellular compartments during and following polyprotein processing.Because of its enzymatic activities and its critical role in viral replication and polyprotein processing, NS3 constitutes a promising drug target for antiviral therapy (31). NS3pro (residues 1 to 169) is a trypsin-like serine protease with the characteristic catalytic triad (Asp-His-Ser) and a highly specific substrate recognition sequence, conserved in all flaviviruses, consisting of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1′ (11). NS3pro has an aberrant fold compared to the canonical trypsin structure, and its folding and protease activity are dependent on a noncovalent association with a central 47-amino-acid hydrophilic domain of NS2B (19, 21). The remainder of NS2B contains three transmembrane helices involved in membrane associations. NS3 mediates cleavages at the C-terminal side of the highly conserved dibasic residue located at the coding junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 and also between the C terminus of C and NS4A (11, 33) (Fig. ​(Fig.11).The C-terminal portion of NS3 (NS3hel, residues 170 to 619) performs several catalytically related activities, namely, RNA strand separation and (poly)nucleotide hydrolysis (5, 22, 32, 55) at a common, RecA-like NTPase catalytic center that couples the energy released from the hydrolysis of the triphosphate moieties of nucleotides to RNA unwinding. Although the precise role of NS3 in replication has not been established, its helicase activity is thought to separate nascent RNA strands from the template strands and to assist replication initiation by unwinding RNA secondary structure in the 3′ untranslated region (11, 13, 15, 33). NS3 is a member of the DEAH/D box family within helicase superfamily 2 (SF2) and is characterized by seven conserved sequence motifs involved in nucleic acid binding and hydrolysis (45). In addition, its RNA triphosphatase activity is thought to be involved in the capping of the viral RNA. In the process of replication, NS3 interacts, most likely via its C-terminal domain, with NS5 (13, 15, 24, 26, 58, 62). The NS3 5′ triphosphatase and NS5 methyltransferase activities probably cooperate in cap formation by removing the terminal γ-phosphate and performing sequential N7 and 2′ O methylations, respectively (16, 28, 46, 56). The guanylyltransferase activity required for cap formation remains elusive at present, although recent evidence suggests that it may be present in NS5 (8, 17). In addition, the interaction between NS3 and NS5 can stimulate NS3 helicase/NTPase activity (15, 62).The atomic structures of NS3pro in the presence and absence of ligands and/or the NS2B activating domain (2, 19, 47) and NS3hel (35, 38, 39, 49, 58-60) are known, and recently, the structure of full-length DENV4 (one of four dengue virus serotypes) NS3 fused to an 18-residue NS2B cofactor (NS2B₁₈NS3) was reported (34). This structure revealed an elongated conformation, with the protease domain interfacing with the NTP binding pocket and being separated from NS3hel by a relatively flexible linker, which suggested that the protease domain may have a positive effect on the activity of the NTPase/helicase domain. However, other reports suggested that NS3pro has no or a very limited effect on the activity of NS3hel (32, 62). In addition, since current evidence suggests that NS2B is not part of the replication complex (Fig. ​(Fig.1)1) (36), and it is known that in the absence of the NS2B cofactor, NS3pro is unfolded and inactive, it becomes hard to envisage what effect the NS3 protease domain may have on the helicase domain in a biologically relevant context. Equally, it is still not clear what role the helicase domain plays during polyprotein processing by NS3pro and, in general, why these two apparently distinct and unrelated catalytic activities are harbored within a single polypeptide.In order to gain further insights into these questions, we report the biochemical analysis and crystallographic structure at a 2.75-Å resolution of full-length NS3 from Murray Valley encephalitis virus (MVEV), a member of the JEV group of flaviviruses, fused to the entire protease activation peptide of the NS2B cofactor (NS2B₄₅NS3). The structure reveals the protease and helicase domains to be structurally independent and differs dramatically from the structure observed for DENV4 NS2B₁₈NS3. We discuss the implications of this unexpectedly different configuration of the NS3 protein and argue that the structural flexibility observed is likely to be crucial for its multifunctional nature.     

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.     

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.