Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells

Purinergic Signalling - Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour...     

Diversity of culturable actinomycetes in hyper-arid soils of the Atacama Desert,Chile

The Atacama Desert presents one of the most extreme environments on Earth and we report here the first extensive isolations of actinomycetes from soils at various locations within the Desert. The use of selective isolation procedures enabled actinomycetes to be recovered from arid, hyper-arid and even extreme hyper-arid environments in significant numbers and diversity. In some cases actinomycetes were the only culturable bacteria to be isolated under the conditions of this study. Phylogenetic analysis and some phenotypic characterisation revealed that the majority of isolates belonged to members of the genera Amycolatopsis, Lechevalieria and Streptomyces, a high proportion of which represent novel centres of taxonomic variation. The results of this study support the view that arid desert soils constitute a largely unexplored repository of novel bacteria, while the high incidence of non-ribosomal peptide synthase genes in our isolates recommend them as promising material in screening for new bioactive natural products.     

The Mature Virion of Ectromelia Virus,a Pathogenic Poxvirus,Is Capable of Intrahepatic Spread and Can Serve as a Target for Delayed Therapy

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.     

Fusion between Perinuclear Virions and the Outer Nuclear Membrane Requires the Fusogenic Activity of Herpes Simplex Virus gB

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic “fusion loops.” These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.Herpesvirus glycoproteins gB and gH/gL participate in two separate membrane fusion events that occur during different stages of virus replication. First, during virus entry into cells, gB and gH/gL promote fusion between the virion envelope and either the plasma membrane or endosomes (reviewed in references 6, 21, 27, and 39). Second, herpes simplex virus (HSV) gB and gH (likely complexed to form a heterodimer with gL), and likely homologues in other herpesviruses, promote nuclear egress (12). Herpesvirus capsids are produced in the nucleus and cross the nuclear envelope (NE) by envelopment at the inner nuclear membrane (NM), producing perinuclear virions that then fuse with the outer NM (reviewed in references 35 and 36). There is evidence that HSV gB and gH/gL function in a redundant fashion in fusion between enveloped, perinuclear virus particles and the outer NM (12), whereas both gB and gH/gL are essential for entry fusion (8, 13, 38). Much more is known about the mechanisms involved in entry fusion than those involved in egress fusion, and many important questions remain in terms of how these two membrane fusion processes relate to each other.Entry of HSV into cells involves interactions between the viral receptor-binding protein gD and the gD receptors (16, 28, 30, 37). When gD binds to its receptors, there are conformational changes in gD which apparently activate gB and gH/gL, so that these glycoproteins promote fusion involving the virion envelope and cellular membranes (21, 32). By using split green fluorescent protein fusion proteins, also denoted bimolecular complementation, two groups showed that gD binding to gD ligands triggers interactions between gB and gH/gL and that this is accompanied by cell-cell fusion (1, 2). There is also evidence that gB and gH/gL contribute to different stages of membrane fusion. When gH/gL is expressed with gD, there is hemifusion (mixing of the outer leaflets of membranes) of adjacent cells, and this partial fusion is apparently mediated by gH/gL (41). However, full fusion (mixing of both inner and outer leaflets) occurs only when gB is coexpressed with gD and gH/gL (41). Also supporting a role for gH in membrane fusion, peptides based on heptad repeats in gH can disrupt model membranes (14, 15, 17). HSV gB is a class III fusion protein, structurally similar to vesicular stomatitis virus G protein, with a three-stranded coil-coil barrel in the central region of the molecule reminiscent of class I fusion proteins, e.g., influenza virus hemagglutinin (22). Therefore, herpesvirus gB and gH/gL differ substantially from the fusion proteins expressed by all other well-studied viruses because both gB and gH/gL participate directly in membrane fusion, apparently functioning in different aspects of entry fusion.HSV gB and other viral class III fusion proteins differ from class I fusion proteins that have N-terminal, hydrophobic fusion peptides because class III fusion proteins possess internal bipartite “fusion loops” composed of both hydrophobic and hydrophilic residues (3, 22). In the solved structure of the HSV gB ectodomain, which might represent a postfusion form of the protein, the fusion loops are located near the base of the molecule, adjacent to the virion envelope (22). Mutant forms of gB with single amino acid substitutions in these fusion loops displayed diminished cell-cell fusion activity when transfected into cells with gD and gH/gL (20). Cell-cell fusion approximates the fusion that occurs during entry, defining the minimal fusion machinery, although there are differences between entry and cell-cell fusion (10). Moreover, full-length gB molecules with fusion loop mutations failed to complement gB null HSV (19). Recently, it was demonstrated that the HSV gB extracellular domain can interact with liposomes in vitro and that this binding depends upon gB''s fusion loops (19).Herpesvirus capsids are assembled in the nucleus and acquire an envelope by budding through the inner NM. For a short time, enveloped virus particles are found in the space between the inner and outer NMs (perinuclear space), but then the envelopes of these particles fuse with the outer NM, releasing capsids into the cytoplasm (reviewed in references 35 and 36). Cytoplasmic capsids acquire a second envelope by budding into the trans-Golgi network, and this secondary envelopment involves redundant or additive functions of gE/gI and gD, i.e., either of these glycoproteins will suffice (11). The second step of the nuclear egress pathway involving membrane fusion between the envelope of perinuclear particles and the outer NM requires HSV glycoproteins gB and gH/gL (12). HSV double mutants lacking both gB and gH accumulate enveloped virus particles in the perinuclear space and in herniations, i.e., membrane vesicles that bulge into the nucleoplasm and derive from the inner NM (12). These observations, coupled with the evidence that gB and gH/gL are fusion proteins, suggested that gB and gH/gL promote the fusion between virus particles and the outer NM. However, there is one important difference between nuclear egress fusion and entry fusion. Virus mutants lacking either gB or gH are unable to enter cells, but such mutants have fewer defects in nuclear egress than double mutants lacking both gB and gH (12). Thus, as with secondary envelopment that involves gD and gE/gI, glycoproteins gB and gH/gL act in a redundant or additive fashion to mediate the fusion between the envelope of perinuclear virus particles and the outer NM. It is also important to note that there appear to be other mechanisms by which HSV particles can exit the perinuclear space. For example, although a substantial number of gB gH null double mutants accumulated in herniations (increased by ∼10-fold), some virions were seen on cell surfaces, although their numbers were reduced by ∼2.5- to 5-fold compared with those of wild-type HSV (12, 46).HSV entry fusion is triggered by gD binding to one of its ligands. However, it is not clear what triggers fusion of the envelope of perinuclear particles with the outer NM. gD, gB, gH, gM, gK, and other viral membrane proteins are all present in NMs and in perinuclear virus particles (4, 12, 25, 40, 42, 44). It seems unlikely that there are substantial quantities of known gD receptors in NMs, although this has not been carefully examined and there may well be unidentified gD receptors present in NMs. However, if fusion at NMs is not activated by gD binding to gD receptors, there must be other mechanisms to trigger this fusion. There is evidence that HSV gK negatively regulates fusion at the NE because (i) overexpression of gK causes enveloped virus particles to accumulate in the perinuclear space (25) and (ii) gK is primarily localized to the endoplasmic reticulum and NM and is not substantially found in extracellular virions (26, 34). Another potential regulatory mechanism for fusion at the outer NM involves phosphorylation of the cytoplasmic domain of gB by the HSV kinase US3 (46). An HSV recombinant lacking gH and expressing a mutant gB with a substitution, T887A, affecting an amino acid in the gB cytoplasmic domain displayed reduced US3-dependent phosphorylation and accumulated enveloped virus particles in herniations (46). This mutation in gB did not alter HSV entry into cells (31, 46). Together, these results suggest that HSV fusion with the outer NM differs from entry fusion in some, but likely not all, important mechanistic details.Given that both gB and gH/gL are well established as fusion proteins for virus entry, we hypothesized that these glycoproteins directly mediate the membrane fusion that occurs between the envelope of perinuclear virus particles and the outer NM (12, 46). However, there are other possibilities. For example, it is conceivable that loss of both gB and gH alters the structure of the envelope of perinuclear HSV virions so that other HSV glycoproteins (that directly promote fusion) are affected. To address this issue and extend our understanding of how gB functions in nuclear egress fusion, we constructed HSV recombinants that express mutant forms of gB with substitutions in the fusion loops. These viruses also lacked gH, making nuclear egress totally dependent on a functional form of gB. By propagating these recombinants using gH-expressing cells, we could produce virus particles including gH and the mutant gB molecules. These HSV recombinants expressing gH as well as gB fusion loops, W174R, W174Y, Y179K, and A261D, were all unable to enter cells. However, two recombinants, expressing W174Y and Y179K, exhibited some cell-to-cell spread while the other two, expressing W174R and A261D, did not spread beyond single infected cells. All four recombinants infected into cells lacking gH exhibited defects in nuclear egress. These results provide strong support for the hypothesis that gB acts directly to mediate the fusion of the virion envelope with the outer NM during HSV egress.     

Capturing the herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment

Herpes simplex virus (HSV) entry requires the core fusion machinery of gH/gL and gB as well as gD and a gD receptor. When gD binds receptor, it undergoes conformational changes that presumably activate gH/gL, which then activates gB to carry out fusion. gB is a class III viral fusion protein, while gH/gL does not resemble any known viral fusion protein. One hallmark of fusion proteins is their ability to bind lipid membranes. We previously used a liposome coflotation assay to show that truncated soluble gB, but not gH/gL or gD, can associate with liposomes at neutral pH. Here, we show that gH/gL cofloats with liposomes but only when it is incubated with gB at pH 5. When gB mutants with single amino acid changes in the fusion loops (known to inhibit the binding of soluble gB to liposomes) were mixed with gH/gL and liposomes at pH 5, gH/gL failed to cofloat with liposomes. These data suggest that gH/gL does not directly associate with liposomes but instead binds to gB, which then binds to liposomes via its fusion loops. Using monoclonal antibodies, we found that many gH and gL epitopes were altered by low pH, whereas the effect on gB epitopes was more limited. Our liposome data support the concept that low pH triggers conformational changes to both proteins that allow gH/gL to physically interact with gB.     

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.Entry of herpes simplex virus (HSV) into cells requires four viral glycoproteins, gB, gD, gH, and gL, plus one of several cell receptors, either herpesvirus entry mediator (HVEM), nectin-1, or 3-OST (45). Crystal structures and other studies have documented that receptor binding triggers conformational changes to gD that trigger the downstream events leading to fusion (10, 11, 18, 26, 28, 52). Moreover, when HSV receptor-bearing cells are transfected with expression plasmids for glycoproteins gB, gD, gH, and gL, the cells fuse to form multinucleated giant cells or syncytia (39, 48). However, the precise series of events that take place after receptor binding have not yet been fully elucidated. What we do know is that both gB and a heterodimer of gH/gL constitute the core fusion machinery that is conserved and required for the fusion step of entry of all herpesviruses (18, 26, 30, 46, 49).Thus far, we know the crystal structure of one form of the gB ectodomain of HSV type 1 (HSV-1) (19). This protein has the characteristics of a fusion protein and is a charter member of the class III group of viral fusion proteins (4). Others in this class include Epstein-Barr virus gB, vesicular stomatitis virus (VSV) G, and baculovirus gp64 (5, 22, 41). Like VSV G and gp64, gB has two putative fusion loops at the base of each protomer of the crystallized trimer. Single-amino-acid mutations in many of the hydrophobic residues of the putative fusion loops of gB ablate its ability to function in cell-cell fusion assays (16, 17). Moreover, these mutants are unable to complement the entry of a gB-null virus (16). Finally, the ectodomains of these mutants, unlike wild-type protein, failed to coassociate with liposomes, indicating that the putative fusion loops do insert into membranes (16, 17). Recently, it was shown that several of these mutants are also defective for fusion events involved in virus egress (51). Together, these studies provide compelling evidence that HSV gB functions as a fusion protein and that the fusion loops are critical for this function. However, unlike VSV G and baculovirus gp64, gB does not function on its own in entry but, rather, requires the participation of gH/gL. In the absence of crystallographic data for gH/gL, it is not yet clear what role it plays in herpesvirus fusion. In a previous study, we used bimolecular complementation (BiMC) to examine protein-protein interactions that occur among the viral glycoproteins during fusion (1). A similar study was carried out by Avitabile et al. (2). The BiMC assay is based on the observation that N- and C-terminal fragments of green fluorescent protein (GFP) (and derivatives such as enhanced yellow fluorescent protein [EYFP]) do not spontaneously reconstitute a functional fluorophore (20, 29, 40). However, the codons for each half can be appended to the genes for two interacting proteins (23, 24). When these are cotransfected, an interaction between the two proteins of interest brings the two halves of the fluorophore in close enough contact to restore fluorescence.When HSV receptor-bearing cells, such as B78H1 cells that are engineered to express nectin-1, are transfected with plasmids that express gB, gD, gH, and gL, they undergo cell-cell fusion (13, 15, 27, 31, 48). When gD is omitted, no fusion occurs. We found that fusion of these transfected cells could be triggered by addition of a soluble form of gD (the gD ectodomain). We then used this approach to examine interactions between gB and gH/gL during cell fusion (1). Therefore, we tagged gB with the C-terminal half of EYFP and gH with the N-terminal half. When plasmids bearing these forms were cotransfected into C10 cells along with a plasmid for untagged gL, no fusion occurred, but importantly, no BiMC occurred. However, when we added gD306, cells began to fuse within 10 min, and all of the syncytia that formed exhibited bright EYFP fluorescence indicative of BiMC. We concluded that gD triggers both fusion and a physical interaction between gB and gH/gL. However, these experiments did not separate these two events, so we were unable to determine if the interaction preceded fusion or merely was a by-product of it.The purpose of this study was to determine if the gB-gH/gL interaction is essential for fusion and if it occurs prior to fusion. We focused on gB because its structure is known and we have a panel of well-characterized monoclonal antibodies (MAbs) to gB. Our approach was to determine which of these MAbs, if any, could block fusion and also block the interaction with gH/gL. We also examined the effect of mutations to the fusion loops of gB on its interaction with gH/gL. We previously mapped these MAbs to four functional regions (FR) of gB, three of which were resolved in the crystal structure (6, 19). Of these, FR1 contains the fusion loops, FR2 is in the center of the gB structure with no known function, and FR3 is at in the crown of the protein and may be involved in binding to cells (7). Our rationale was that if the interaction between gB and gH/gL is important for fusion, then it should not be blocked by nonneutralizing anti-gB MAbs. At the same time, we thought that some neutralizing MAbs might not only block fusion but also block BiMC. We found that neutralizing MAbs to FR1 and FR2 inhibited both BiMC and fusion. In contrast, we found that neutralizing MAbs that map to FR3 blocked fusion but failed to block the interaction between gB and gH/gL, thereby dissociating the two events. Finally, we found that gB mutants with changes in the fusion loops that were fusion negative were also unable to bind to gH/gL. The latter results suggest that insertion of gB into the target membrane precedes its interaction with gH/gL.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.