Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

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.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

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 Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.