Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended “flap” masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.     

A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.The lipid bilayer membranes of living cells pose an existential challenge to viruses. In enveloped viruses, viral glycoproteins execute a highly regulated fusion event between virion and cellular membranes, thereby delivering the viral genome and other contents of the virion into the host cell. Antibody responses that block entry are considered neutralizing and represent an important host defense against viral pathogens.In many enveloped viruses, one or two viral glycoproteins suffice to carry out binding and membrane fusion events that mediate entry. In herpesviruses, however, at least four envelope glycoproteins are typically involved. The core machinery for herpesvirus entry comprises three highly conserved viral glycoproteins, glycoprotein B (gB), glycoprotein H (gH), and glycoprotein L (gL), along with one or more accessory glycoproteins necessary for binding to cell surface receptors (reviewed in refs. 1, 2). gB is thought to be the proximal mediator of membrane fusion, whereas gH and gL form a complex, termed gH/gL, which has been found to regulate the fusogenic activity of gB (3–6). In a number of beta and gamma herpesviruses, including the human pathogens human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and Epstein–Barr virus (EBV), two different gH/gL complexes are found on the virion envelope and are necessary for the viruses to enter the full range of cell types that they infect in vivo.Of the two gH/gL complexes expressed in HCMV virions, the gH/gL complex with glycoprotein O (gO), gH/gL/gO, suffices for entry into fibroblasts, a cell type in which fusion events at the plasma membrane initiate infection (7). Infection of several other types of cells, including monocytes, dendritic cells, endothelial cells, and epithelial cells, requires the pentameric complex of gH/gL and three small glycoproteins—UL128, UL130, and UL131 (UL128-131)—and appears to involve fusion at endosomal membranes (8–16). Strains of HCMV, such as AD169 and Towne, that have undergone extensive serial passage in cultured fibroblasts fail to express the pentameric gH/gL/UL128-131 complex on virions and thus are unable to infect epithelial and endothelial cells (12, 13, 15); however, repair of a frameshift mutation in the UL131 gene of strain AD169 restores expression of gH/gL/UL128-131 (11, 12) and expands its cell tropism.Less extensively passaged HCMV strains that retain expression of gH/gL/UL128-131 can efficiently infect epithelial and endothelial cells (13, 17, 18). Nonetheless, several such strains replicate to ∼1,000-fold lower titers on epithelial cells compared with strain AD169 repaired for UL131 (11). AD169 lacks a ∼15-kb region at the end of the unique long genome region, termed the ULb′ (19). We were intrigued by the rather striking differences in cell tropism between laboratory strain AD169 repaired for expression of the pentameric gH/gL/UL128-131 complex, and strains, such as TB40/E, that have largely intact ULb′ regions and maintain expression of gH/gL/UL128-131. We hypothesized the ULb′ region encodes an additional factor involved in HCMV cell tropism. Our studies addressing this hypothesis led us to identify a new function for UL148, a gene within the ULb′. We found that UL148 encodes an endoplasmic reticulum (ER) resident glycoprotein that influences virion cell tropism by regulating the composition of alternative gH/gL complexes.     

Hodgkin's lymphoma, Epstein-Barr virus reactivation and fatal haemophagocytic syndrome

Haemophagocytic syndrome is a serious disorder, often related to Epstein-Barr virus (EBV) or other infectious agents. Frequently an underlying immune abnormality or a T-cell lymphoma is present. The combination of haemophagocytosis and Hodgkin's lymphoma seems to be rare. A 70-year-old female with rheumatoid arthritis was admitted with constitutional symptoms, persistent fever, pancytopenia, deranged liver enzymes, lymphadenopathy and splenomegaly. A fatal coagulopathy supervened. The clinical picture and the bone marrow findings indicated a haemophagocytic syndrome and a lymph node biopsy disclosed an EBV-positive Hodgkin's lymphoma. EBV serology pointed at viral reactivation and a high EBV DNA content was detected in serum by real-time quantitative PCR analysis (5.5 x 10(6) copies per mL). The case history is presented and the literature is reviewed.     

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-ΔP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-ΔY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.     

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy

The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. We developed an experimental system that uses liposomes as the target membrane to capture Sindbis virus, a prototypical alphavirus, in its membrane-binding form at pH 6.4. Cryoelectron micrograph analyses and 3D reconstructions showed that the virus retains its overall icosahedral structure at this mildly acidic pH, except in the membrane-binding region, where monomeric E1 associates with the target membrane and the E2 glycoprotein retains its original trimeric organization. The remaining E2 trimers may hinder E1 homotrimerization and are a potential target for antiviral drugs.     

Paramyxovirus fusion and entry: multiple paths to a common end

The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens.     

Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition

HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼−23 k_BT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.The HIV-1 glycoprotein 41 (gp41) homotrimer constitutes the transmembrane stem of the envelope glycoprotein complex (Env) and plays key roles in viral entry, the first step of viral infection (1–3) (Fig. 1A). Comprising three gp120/gp41 heterodimers, the Env complex recognizes receptors on target cells and primes gp41 for membrane fusion (4, 5). Gp41 is initially held in a largely unfolded state and shielded by gp120 in the metastable Env complex like a loaded spring (1). During membrane fusion, gp41 inserts its fusion peptide into the host cell membrane with the help of gp120, forming an extended gp41 prehairpin conformation (6, 7) (Fig. 1B). Then the extended gp41 folds back, drawing the two membranes into proximity. In this process, gp41 uses its folding energy to lower the energy barrier of membrane fusion and thus increases the rate of fusion. After fusion, gp41 forms an extraordinarily stable trimer-of-hairpins in a six-helix bundle (6HB) conformation, in which three N-terminal heptad repeats (NHRs) form a central coiled coil and three C-terminal heptad repeats (CHRs) bind in the grooves of the NHR coiled coil in an antiparallel fashion (8, 9) (Fig. 1 B and C). The 6HB further extends to the membrane-proximal external region (MPER) and its complementary fusion peptide-proximal region (FPPR) (10). Although significant progress has been made by defining gp41 structures in the initial and final stages of membrane fusion, how gp41 transits between the two structures remains unclear. Addressing this question requires better determination of the intermediates, energy, and kinetics associated with gp41 folding.Open in a separate windowFig. 1.Sequences, conformations, and constructs of gp41 and the experimental setup. (A) Domain structure of HIV-1 gp41. The three truncated gp41 sequences used in this study, the sequence of T20, and T20-resistant mutation (N554D) are indicated. CHR, C-terminal heptad repeat; CT, cytoplasmic tail; FP, fusion peptide; FPPR, fusion peptide-proximal region; MPER, membrane-proximal external region; NHR, N-terminal heptad repeat; TMD, transmembrane domain. (B) Diagram showing gp41 folding and viral membrane fusion. In the prehairpin state, three gp41 monomers form a stable three-helix bundle in their NHRs with largely unfolded CHRs. We refer to the conformational transition from this prehairpin state to the six-bundle (6HB) state as gp41 folding, in contrast to de novo formation of the 6HB from free gp41 monomers as measured in previous ensemble experiments (18). (C) Experimental setup to pull a single gp41 complex using dual-trap optical tweezers. (D) Topology of the construct used to pull the third hairpin in the gp41 complex. Two constructs with different loop sizes were made, gp41-ll with a 19-aa loop and gp41-sl with a 5-aa loop. (E) Topology of the construct to specifically pull the second gp41 hairpin. The construct contained a cleavage site of the TEV protease (indicated by x). After TEV cleavage and purification, the C-terminal pulling site of gp41-ll-hp2 was generated.Knowledge of gp41 folding is crucial to understanding the mechanisms of viral membrane fusion and infection as well as pharmaceutical intervention in AIDS (11, 12). Membrane fusion needs to overcome an energy barrier over ∼50 k_BT (13). A commensurate energy is demanded from folding of one or multiple gp41 complexes to catalyze viral membrane fusion. However, a large gp41 folding energy may also destabilize the metastable Env complex (6, 14). Mutations that decrease or increase gp41 stability in turn stabilize or destabilize the Env complex (15). Thus, the folding energy of gp41 seems to be optimized to balance the rate of membrane fusion and the stability of the Env complex, thereby achieving highest infectivity. However, the folding energy of a single gp41 trimer has not been accurately measured owing to irreversibility and protein aggregation associated with gp41 folding in traditional ensemble-based experiments (16).Once attached to the host cell, an HIV virion requires at least 15 min to fuse (6, 17). Several gp120-dependent gp41 folding intermediates have been implicated in the fusion process (6, 7). After gp120 is shed, folding intermediates of the gp41 complex alone have not been reported (18). However, intermediates may appear due to independent or cooperative folding of three gp41 monomers. All these intermediates, regardless of their dependence on gp120, are important, because they are essential for membrane fusion and primary targets of anti-HIV drugs, including various fusion inhibitors and broadly neutralizing antibodies (11, 12). In contrast, the conserved gp41 and gp120 regions are protected by gp120 glycan shield on the surface of the Env complex, and thus are not accessible for most drugs and antibodies (1, 2). T20 (enfuvirtide) is the first commercial antiretroviral fusion inhibitor (19, 20). It is a 36-aa synthetic polypeptide that shares its sequence with regions of CHR and MPER in gp41 (Fig. 1A). Thus, T20 competes with these regions to bind their cognate sequences on gp41 to inhibit gp41 folding (Fig. 1B). T20 tightly associates with the five-helix bundle (5HB) missing one CHR with a dissociation constant around 30 nM and efficiently inhibits HIV-1 viral infection (21). However, the efficacy of T20 is compromised by certain gp41 mutations. Gp41 mutation N554D (or N43D based on gp41 amino acid numbering) is a common T20-resistant mutation identified from AIDS patients treated with T20 (22). How T20 efficiently inhibits folding of the wild-type, but not the drug-resistant, gp41 is not well understood.Gp41 folding is profoundly affected by membranes. Several studies showed that gp41 fails to fold into the 6HB conformation in the presence of membranes or detergent micelles but remains in largely unfolded monomer form (23, 24). However, other studies suggested that gp41 does form the 6HB structure that can even be strengthened by SDS (25). These different reported affinities between gp41 monomers and membranes or membrane mimics inversely correlate with the stability of the 6HB and lead to different models of membrane fusion. In principle, proteins that mediate membrane fusion, including viral fusion proteins and SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins, may promote membrane fusion either mechanically or chemically. In the mechanical model, fusion proteins, such as SNARE proteins, transduce their folding energy to mechanical force to draw two membranes into proximity for fusion (26). A single neuronal SNARE complex folds/assembles in multiple stages and outputs a total energy of −65 k_BT (27), or −41 kcal/mol. In the chemical model, fusion proteins or chemical agents such as polyethylene glycol alter membrane properties (for example, by dehydration of membranes) to enhance membrane merging without large energy output from folding of fusion proteins, much like strewing salt on snow. However, these two extreme models may not be exclusive. It will be interesting to see whether gp41 fuses membranes similarly to SNARE proteins or by a different mechanism.Finally, HIV-1 gp41 represents a family of structurally and mechanistically conserved class-I viral fusion proteins, including the glycoprotein GP2 of Ebola virus and hemagglutinin of the influenza virus (1, 4). To our knowledge, none of these proteins has been well studied in terms of their folding energy and kinetics. Therefore, detailed measurements of gp41 folding energy and kinetics will shed light on the working mechanism of these viral fusion proteins and help to develop antiretroviral fusion inhibitors against diseases caused by these viruses (11, 20).We investigated folding of a single gp41 complex using high-resolution optical tweezers (28). Our single-molecule manipulation approach used mechanical force to unfold the gp41 complex under a physiological solution condition, to probe its folding energy and kinetics, and to mimic the membrane repulsive force during functional gp41 folding. In addition, our method overcame ensemble averaging and synchronization required by traditional protein folding studies that often obscure folding intermediates (27, 29). We found that each gp41 hairpin in the complex folded in a two-state manner and kinetically coupled with other hairpins. The total folding energy of a single gp41 complex was estimated to be −71 k_BT. We discovered that T20 inhibited gp41 folding by a novel force-dependent strand-displacement mechanism. Although detergent micelles destabilize the gp41 complex, they do not abolish the complex formation.     

Single nucleotide insertion in the 5′‐untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfection

Abstract: Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world‐wide. Antiviral therapy‐related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV‐related liver cirrhosis, but the role of the 5′‐untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5′‐UTR in an HCV‐infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5′‐UTR of HCV showed a nucleotide A insertion at position 193 of the IRES.     

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM To study the epitope distribution of hepatitis G virus(HGV) and to seek for the potential recombinant antigensfor the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV genefragments from core to NS3 and NS5 were constructedusing prokaryotic expression vector pRSET and (or)pGEX,and expressed in E.coli.Western blotting andELISA were used to detect the immunoreactivity of theserecombinant proteins.RESULTS One clone with HGV fragment from core to E1(G1),one from E2 (G31),three from NS3 (G6,G61,G7),one from NS5B (G821) and one chimeric fragment fromNS3and NS5B (G61-821) could be expressed well andshowed obvious immunoreactivity by Western blotting.One clone with HGV framment from NSSB (G82) was alsowell expressed,but could not show immunoreactivity byWestern blotting.No obvious expression was found in theother six clones.All the expressed recombinant proteinswere in inclusion body form,except the protein G61 whichcould be expressed in soluble form.Further purifiedrecombinant proteins G1,G31,G61,G821 and G61-821were detected in indirected ELISA as coating antigenrespectively.Only recombinant G1 could still showimmunoreactivity,and the other four recombinantproteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivityof these four recombinant proteins were lost duringpurification.CONCLUSION Core to E1,E2,NS3 and NS5 fragment ofHGV contain antigenic epitopes,which could be producedin prokaryotically expressed recombinant proteins.Ahigh-yield recombinant protein (G1) located in HGV coreto E1 could remain its epitope after purification,whichshowed the potential that G1 could be used as a coatingantigen to develop an ELISA kit for HGV specific antibodydiagnosis.     

Ganciclovir and the treatment of Epstein-Barr virus hepatitis

Epstein-Barr virus (EBV) is part of the herpesvirus family that infects up to 90% of the population. Initial infection is often subclincal in children but will generally result in symptomatic infectious mononucleosis in adolescents and adults. Ganciclovir has been utilized in immunocompromised patients with EBV encephalitis and post-liver transplant for EBV fulminant hepatitis. Herein, the successful use of ganciclovir in two immunocompetent patients with severe EBV hepatitis is reported.     

From the Cover: Identification of transferrin receptor 1 as a hepatitis C virus entry factor

Hepatitis C virus (HCV) is a liver tropic pathogen that affects ∼170 million people worldwide and causes liver pathologies including fibrosis, cirrhosis, steatosis, iron overload, and hepatocellular carcinoma. As part of a project initially directed at understanding how HCV may disrupt cellular iron homeostasis, we found that HCV alters expression of the iron uptake receptor transferrin receptor 1 (TfR1). After further investigation, we found that TfR1 mediates HCV entry. Specifically, functional studies showed that TfR1 knockdown and antibody blocking inhibit HCV cell culture (HCVcc) infection. Blocking cell surface TfR1 also inhibited HCV pseudoparticle (HCVpp) infection, demonstrating that TfR1 acts at the level of HCV glycoprotein-dependent entry. Likewise, a TfR1 small-molecule inhibitor that causes internalization of surface TfR1 resulted in a decrease in HCVcc and HCVpp infection. In kinetic studies, TfR1 antibody blocking lost its inhibitory activity after anti-CD81 blocking, suggesting that TfR1 acts during HCV entry at a postbinding step after CD81. In contrast, viral spread assays indicated that HCV cell-to-cell spread is less dependent on TfR1. Interestingly, silencing of the TfR1 trafficking protein, a TfR-1 specific adaptor protein required for TfR1 internalization, also inhibited HCVcc infection. On the basis of these results, we conclude that TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization.     

Heterogeneous expression of Epstein-Barr virus latent proteins in endemic Burkitt's lymphoma

Epstein-Barr virus (EBV)-infected cells may sustain three distinct forms of virus latency. In lymphoblastoid cell lines, six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B), and two nuclear RNAs (EBERs) are expressed. This form of latency, termed latency III, is also encountered in some posttransplant lymphoproliferative disorders. In EBV-positive cases of Hodgkin's disease, the EBERs, EBNA1, and the LMPs are expressed (latency II), whereas in Burkitt's lymphoma (BL) only the EBERs and EBNA1 have been detected (latency I). We have studied the expression of EBV proteins in 17 cases of EBV-positive endemic BL by immunohistology. Expression of LMP1 was seen in variable proportions of tumor cells in two cases and EBNA2 was detected in some tumor cells in three other cases. Also, the BZLF1 trans-activator protein was expressed in a few tumor cells in 6 cases, indicating entry into the lytic cycle. A phenotypic drift from latency I to latency III has been observed previously in some BL cell lines. Our results suggest that a similar phenomenon may occur in BL in vivo and indicate that the operational definition of EBV latencies is not easily applied to human tumors.     

CD134 is a cellular receptor specific for human herpesvirus-6B entry

Human herpesvirus-6B (HHV-6B) is a T lymphotropic β-herpesvirus that is clearly distinct from human herpesvirus-6A (HHV-6A) according to molecular biological features. The International Committee on Taxonomy of Viruses recently classified HHV-6B as a separate species. The primary HHV-6B infection causes exanthem subitum and is sometimes associated with severe encephalopathy. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection. HHV-6B reactivation causes encephalitis in immunosuppressed patients. The cellular receptor for HHV-6A entry was identified as human CD46, but the receptor for HHV-6B has not been clear. Here we found that CD134, a member of the TNF receptor superfamily, functions as a specific entry receptor for HHV-6B. A T-cell line that is normally nonpermissive for HHV-6B infection became highly susceptible to infection when CD134 was overexpressed. CD134 was down-regulated in HHV-6B–infected T cells. Soluble CD134 interacted with the HHV-6B glycoprotein complex that serves as a viral ligand for cellular receptor, which inhibited HHV-6B but not HHV-6A infection in target cells. The identification of CD134 as an HHV-6B specific entry receptor provides important insight into understanding HHV-6B entry and its pathogenesis.     

Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants.

Transformed cell lines derived from murine C127 cells were constructed that express the influenza virus RNA-dependent RNA polymerase proteins (PA, PB1, and PB2). Cell lines that express only one or all three of the proteins were tested for their ability to complement temperature-sensitive viral mutants incubated at the nonpermissive temperature. Two cell lines were isolated that express all three polymerase genes and complement the growth of PB2 temperature-sensitive mutants at the nonpermissive temperature. One of these lines also complemented PA temperature-sensitive mutants. The viral titers obtained in these two cell lines were 12-fold to 1000-fold higher than the viral titers obtained upon growth of the corresponding temperature-sensitive mutant in C127 cells at the nonpermissive temperature.     

从病毒性心肌炎演变为扩张型心肌病:病毒的作用

病毒感染、宿主免疫反应、以及遗传和环境变化是决定病毒性心肌炎向扩张型心肌病演变的重要因素,其中病毒感染既是启动又是影响疾病发生发展的关键环节。近年研究发现,病毒不仅对心肌细胞有直接和间接损伤作用,而且还通过逃逸宿主先天免疫、诱导免疫因子分泌或表达异常等机制推动疾病进程。抗病毒治疗有益于病毒性心肌炎患者的恢复,在一定程度上抑制扩张型心肌病的发生。     

罕见成人慢性活动性EB病毒感染1例报告

正1病例资料患者男性,27岁,因"反复发热7周,发现肝功能异常1个月"于2014年8月20日入本院。患者于7周前无明显诱因出现发热,多发生于晚间(约16∶00~19∶00),体温最高为38.5℃,伴有咽痛,偶有咳嗽、咳痰,痰为白色泡沫样,伴有散在皮肤疱疹,约米粒大小,自服"布洛芬"后体温逐渐降至正常,不曾系     

Filovirus entry: a novelty in the viral fusion world

Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of this entry pathway are highlighted here. Virus entry into cells is initiated by the interaction of the viral glycoprotein(1) subunit (GP(1)) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP(1) is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP(1) are currently incomplete; however, the processed GP(1) specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP(1) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP(2) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.     

Mechanisms of coronavirus cell entry mediated by the viral spike protein

Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes-A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.     

Myelin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are prevalent neurotropic herpesviruses that cause various nervous system diseases. Similar to other enveloped viruses, membrane fusion is an essential process for viral entry. Therefore, identification of host molecules that mediate membrane fusion is important to understand the mechanism of viral infection. Here, we demonstrate that myelin-associated glycoprotein (MAG), mainly distributed in neural tissues, associates with VZV glycoprotein B (gB) and promotes cell-cell fusion when coexpressed with VZV gB and gH/gL. VZV preferentially infected MAG-transfected oligodendroglial cells. MAG also associated with HSV-1 gB and enhanced HSV-1 infection of promyelocytes. These findings suggested that MAG is involved in VZV and HSV infection of neural tissues.