共查询到20条相似文献,搜索用时 46 毫秒
1.
Kelly K Lee 《The EMBO journal》2010,29(7):1299-1311
Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three‐dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo‐tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane‐centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky’ fusion to the observed prefusion structures is discussed. 相似文献
2.
Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins 总被引:6,自引:0,他引:6
Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity. 相似文献
3.
The structure of a membrane fusion mutant of the influenza virus haemagglutinin. 总被引:4,自引:0,他引:4
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH. 相似文献
4.
Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion. 总被引:8,自引:8,他引:0
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles. 相似文献
5.
Influenza viruses were able to mediate fusion of DNA-loaded liposomes with living cultured cells such as monkey COS-7 cells. This was inferred from the appearance of CAT activity in recipient cells incubated with the combination of influenza viruses and liposomes loaded with the plasmid pSV2CAT. Influenza virions were found to be as efficient as intact Sendai virions in mediating microinjection of foreign DNA into living cells. Also, reconstituted envelopes bearing either influenza glycoproteins or the combination of Sendai and influenza glycoproteins were highly efficient in promoting fusion of loaded liposomes with recipient cells. Introduction of DNA into cultured cells required the presence of an active influenza fusion protein; namely, an active HA glycoprotein. Very little or no CAT activity was observed in cells incubated with loaded liposomes and unfusogenic influenza viruses. The virus-induced fusion event probably occurs within intracellular organelles such as endosomes following receptor-mediated endocytosis of virus-liposome complexes. This is due to the fact that the viral fusion glycoprotein is activated only at acidic pH values such as those which characterize the intraendosomal environment. Results of the present work demonstrate for the first time microinjection of foreign DNA via fusion with membranes of intracellular organelles. The potential of the present system to serve as a biological carrier for in vivo use is discussed. 相似文献
6.
Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection. 相似文献
7.
Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《The Journal of general physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane. 相似文献
8.
Sougrat R Bartesaghi A Lifson JD Bennett AE Bess JW Zabransky DJ Subramaniam S 《PLoS pathogens》2007,3(5):e63
The envelope glycoproteins of primate lentiviruses, including human and simian immunodeficiency viruses (HIV and SIV), are heterodimers of a transmembrane glycoprotein (usually gp41), and a surface glycoprotein (gp120), which binds CD4 on target cells to initiate viral entry. We have used electron tomography to determine the three-dimensional architectures of purified SIV virions in isolation and in contact with CD4+ target cells. The trimeric viral envelope glycoprotein surface spikes are heterogeneous in appearance and typically approximately 120 A long and approximately 120 A wide at the distal end. Docking of SIV or HIV-1 on the T cell surface occurs via a neck-shaped contact region that is approximately 400 A wide and consistently consists of a closely spaced cluster of five to seven rod-shaped features, each approximately 100 A long and approximately 100 A wide. This distinctive structure is not observed when viruses are incubated with T lymphocytes in the presence of anti-CD4 antibodies, the CCR5 antagonist TAK779, or the peptide entry inhibitor SIVmac251 C34. For virions bound to cells, few trimers were observed away from this cluster at the virion-cell interface, even in cases where virus preparations showing as many as 70 envelope glycoprotein trimers per virus particle were used. This contact zone, which we term the "entry claw", provides a spatial context to understand the molecular mechanisms of viral entry. Determination of the molecular composition and structure of the entry claw may facilitate the identification of improved drugs for the inhibition of HIV-1 entry. 相似文献
9.
The three-dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion-competent (low pH) form by electron cryo-microscopy at a resolution of 10 A and 14 A, respectively, have been determined. In the fusion-competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so-called fusion sequence of HA towards the target membrane. 相似文献
10.
Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the postfusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed, larger particles representing fused virions appear, the HA spikes become conspicuously disorganized, a layer of M1 matrix protein is no longer resolved on most virions, and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, respectively. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH. 相似文献
11.
Libersou S Albertini AA Ouldali M Maury V Maheu C Raux H de Haas F Roche S Gaudin Y Lepault J 《The Journal of cell biology》2010,191(1):199-210
The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH-induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction. 相似文献
12.
Xu Zhang Tim Abel Shuo Su Andreas Herrmann Kai Ludwig Michael Veit 《The Journal of biological chemistry》2022,298(3)
Assembly and budding of the influenza C virus is mediated by three membrane proteins: the hemagglutinin-esterase-fusion glycoprotein (HEF), the matrix protein (CM1), and the ion channel (CM2). Here we investigated whether the formation of the hexagonal HEF arrangement, a distinctive feature of influenza C virions is important for virus budding. We used super resolution microscopy and found 250-nm sized HEF clusters at the plasma membrane of transfected cells, which were insensitive to cholesterol extraction and cytochalasin treatment. Overexpression of either CM1, CM2, or HEF caused the release of membrane-enveloped particles. Cryo-electron microscopy of the latter revealed spherical vesicles exhibiting the hexagonal HEF clusters. We subsequently used reverse genetics to identify elements in HEF required for this clustering. We found that deletion of the short cytoplasmic tail of HEF reduced virus titer and hexagonal HEF arrays, suggesting that an interaction with CM1 stabilizes the HEF clusters. In addition, we substituted amino acids at the surface of the closed HEF conformation and identified specific mutations that prevented virus rescue, others reduced virus titers and the number of HEF clusters in virions. Finally, mutation of two regions that mediate contacts between trimers in the in-situ structure of HEF was shown to prevent rescue of infectious virus particles. Mutations at residues thought to mediate lateral interactions were revealed to promote intracellular trafficking defects. Taken together, we propose that lateral interactions between the ectodomains of HEF trimers are a driving force for virus budding, although CM2 and CM1 also play important roles in this process. 相似文献
13.
While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition, the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested. 相似文献
14.
Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes 总被引:3,自引:0,他引:3
We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein. 相似文献
15.
Ciampor F 《Acta microbiologica et immunologica Hungarica》2003,50(4):433-442
Pathogenicity and virulence are multifactorial traits, depending on interaction of viruses with susceptible cells and organisms. The ion channels coded by viruses, viroporins, represent only one factor taking part in the cascade of interactions between virus and cell, leading to the entry of virus, replication and to profound changes in membrane permeability. The M2 protein from influenza A virus forms proton-selective, pH-regulated channel involved in regulating vesicular pH, a function important for the correct maturation of HA glycoprotein. The NB glycoprotein of influenza B viruses is an integral membrane protein with an ion channel activity. The CM2 protein of influenza C virus is an integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. The picornavirus 3A protein is involved in cell lysis and shows homology with other lytic proteins. Vpu is an oligomeric integral membrane protein encoded by HIV-1, which forms ion channels. The togavirus 6K protein shows structural similarities with other viroporins. 相似文献
16.
S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA. 相似文献
17.
One of the key stages of cell infection with influenza virus is the enveloped virus fusion with the cell endosome membrane. To study fusion of single fluorescently-labeled influenza virions with a model bilayer membrane (BLM), a special model system was developed. A small patch of BLM with several adsorbed virions was localized upon a contact with a glass micropipette. Low pH of solution inside the pipette triggered fusion that could be registered by a change in the conductance and integral fluorescence of the BLM patch. It has been shown that the fusion initiation is followed by an increase of fluorescence signal due to the probe redistribution from the virus membrane to the BLM fragment. The increase in fluorescence was accompanied by changes in conductance. Usually, from two to five periods of the channel activity were observed, each of which probably corresponded to fusion of a single virion. It has been found that electric activity was completely inhibited by amantadine known as a blocking agent of M2 channels. This allows one to suggest that the observed changes in conductance are connected with the activity of M2 channels in the virus membrane, whose electric accessibility was the result of fusion of single virions with BLM. 相似文献
18.
Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes 总被引:4,自引:0,他引:4
Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis. 相似文献
19.
The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2. 相似文献
20.
A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range. 总被引:13,自引:4,他引:9
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus. 相似文献