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1.
Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells. 总被引:18,自引:15,他引:3
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We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism. 相似文献
2.
Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.
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The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport. 相似文献
3.
The SU and TM envelope protein subunits of bovine leukemia virus are linked by disulfide bonds, both in cells and in virions
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After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions. 相似文献
4.
Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions. 总被引:53,自引:45,他引:8
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The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant-infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope. 相似文献
5.
Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain. 总被引:1,自引:14,他引:1
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We previously reported that truncation of the cytoplasmic domain of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced its ability to induce cell fusion in a variety of cell lines. In the present study, we examined the expression of the full-length and truncated SIVmac239 envelope glycoprotein complex on cell surfaces. Using a membrane-impermeable reagent to biotinylate proteins on cell surfaces followed by immunoprecipitation, we found that under conditions in which the full-length TM protein could not be detected on the surfaces of CD4-positive or CD4-negative cell lines, the truncated TM protein was detected efficiently. In contrast, using a membrane-impermeable iodination reagent to label proteins on cell surfaces, we could detect both the full-length and truncated TM proteins. No difference between the full-length and truncated proteins was observed in the detection of the SU proteins in the biotinylation assay. Additionally, we used an assay in which SIV-specific antibodies are prebound to the native envelope proteins expressed on the cell surface and then the proteins are immunoprecipitated. Using this assay, we could not detect the truncated or full-length TM protein on the cell surface, whereas we could detect the SU subunits of both proteins. We also observed that the truncated TM protein formed more stable sodium dodecyl sulfate-resistant oligomers than the full-length TM protein did. These results indicate that truncation of the cytoplasmic domain of the SIVmac239 envelope glycoprotein affects the conformation of the external domain of the TM protein on the cell surface, even though the two proteins have no differences in the amino acid sequences of their external domains. This altered conformation could play a role in the enhanced fusion activity of the truncated SIV glycoprotein. 相似文献
6.
New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. 总被引:34,自引:26,他引:8
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We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences. 相似文献
7.
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
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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
8.
A charged amino acid substitution within the transmembrane anchor of the Rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport 总被引:14,自引:0,他引:14
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Two point mutations were introduced by oligonucleotide-directed mutagenesis into the region of the Rous sarcoma virus envelope gene that encodes the hydrophobic transmembrane anchor of the receptor glycoprotein. Single-nucleotide substitutions ultimately converted a hydrophobic leucine, located centrally within the membrane-spanning domain, to either a similarly hydrophobic methionine or a positively charged arginine. The altered coding region was reinserted into an intact copy of the envelope gene, cloned into simian virus 40 late-replacement vector and expressed in primate cells. Analysis of envelope gene expression in CV-1 monkey cells revealed normal levels of synthesis of a membrane-spanning precursor for both the mutants; however, the arginine-containing mutant [mu 26(arg)] exhibited greatly reduced cell surface expression of mature protein, as determined by indirect immunofluorescence and 125I labeling of surface proteins. In experiments in which cells producing the mu 26(arg) polypeptide were pulsed with radioactive leucine and then chased for 5 h, no intracellular accumulation or extracellular secretion of mature products (gp85 and gp37) could be detected. Treatment of mu 26(arg)-infected cells with lysosomal enzyme inhibitors (chloroquine and leupeptin) resulted in the accumulation of gp85 and gp37, indicating that they were being degraded rapidly in lysosomes. The fact that terminally glycosylated and proteolytically cleaved env gene products were observed under these conditions showed that modifications associated with passage through the trans compartment of the Golgi apparatus occurred normally on the mutant polypeptide; thus insertion of a highly charged amino acid into the transmembrane hydrophobic region of gp37 results in the postGolgi transport to lysosomes. It is proposed that the insertion of this mutation into the transmembrane anchor of the envelope glycoprotein does not affect membrane association, orientation with respect to the membrane, or intracellular transport at early stages during maturation. At a step late in the transport pathway, however, the presence of the charged side chain alters the protein in such a manner that the molecules are transported to the lysosomes and degraded. It seems likely that transport of the protein from the trans-Golgi to the cell surface is either directly blocked, or that after expression on the cell surface the mature glycoprotein complex is unstable and rapidly endocytosed. 相似文献
9.
Generation of a water-soluble oligomeric ectodomain of the Rous sarcoma virus envelope glycoprotein. 总被引:8,自引:7,他引:1
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Sequences encoding the transmembrane domain of the Rous sarcoma virus envelope (Env) glycoprotein were deleted and replaced with sequences that signal addition of a glycosyl phosphatidylinositol (GPI) membrane anchor. Stable NIH 3T3 cell lines expressing either the wild-type transmembrane-anchored Env or the Env chimera with a GPI tail were established. The GPI-anchored envelope glycoprotein is expressed, oligomerized, and transported to the cell surface in a manner identical to that of its wild-type transmembrane-anchored counterpart. The GPI-linked protein is quantitatively removed from the cell surface by treatment with phosphatidylinositol phospholipase C. The phosphatidylinositol phospholipase C-released, water-soluble Env glycoprotein ectodomain retains the wild-type oligomeric structure and provides a useful tool for studying the subgroup-specific binding and fusion activities of a prototypic retroviral Env glycoprotein. 相似文献
10.
Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors. 相似文献
11.
trans-activation of the simian virus 40 enhancer by a pX product of human T-cell leukemia virus type I. 总被引:9,自引:3,他引:6
A trans-acting factor, p40, of human T-cell leukemia virus type I profoundly potentiated the function of the enhancer from simian virus 40 but not polyomavirus and Rous sarcoma and murine sarcoma viruses. This trans-activation was seen in a limited repertoire of cells, in contrast to trans-activation of the human T-cell leukemia virus type I enhancer by p40. 相似文献
12.
Retrovirus-expressed hemagglutinin protects against lethal influenza virus infections. 总被引:5,自引:2,他引:3
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An influenza virus hemagglutinin gene, H7, has been expressed in a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. This virus, P1/H7, expressed a glycosylated precursor of the H7 protein which was processed to a mature form and transported to the cell surface. The expressed H7 glycoprotein could not be detected in P1/H7 virus particles. A P1/H7 stock which expressed 5 to 10% of the level of H7 observed in influenza virus-infected chicken embryo fibroblasts was used to immunize 1-month-old chickens. This immunization resulted in low or undetectable levels of hemagglutination-inhibiting and neutralizing antibody. Despite the low serum response, challenge with a highly pathogenic H7N7 virus revealed complete protection against lethal infection. 相似文献
13.
Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells 总被引:2,自引:0,他引:2
Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate. 相似文献
14.
Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.
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Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity. 相似文献
15.
16.
17.
A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells. 总被引:20,自引:15,他引:5
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C C LaBranche M M Sauter B S Haggarty P J Vance J Romano T K Hart P J Bugelski M Marsh J A Hoxie 《Journal of virology》1995,69(9):5217-5227
We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells. 相似文献
18.
19.
Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers. 总被引:2,自引:1,他引:1
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Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors. 相似文献
20.
Chick embryo fibroblasts (CEFs) transformed by Rous sarcoma virus synthesize reduced amounts of fibronectin and also shed this protein into the medium more rapidly than do uninfected cells. We wanted to know whether or not the increased fibronectin shedding rate observed in RSV-transformed CEFs could be explained by an inability of fibronectin to form dimers and/or HMW complex. Our studies on normal and RSV-transformed fibroblasts labelled either metabolically or by lactoperoxidase-catalysed iodination indicate that RSV-induced transformation does not alter the subunit structure of either cell-bound or shed fibronectin, nor does it appear to alter the kinetics of conversion of dimeric fibronectin into HMW complex. We conclude that transformation of CEFs by Rous sarcoma virus does not prevent the assembly of fibronectin into dimeric and HMW complex forms and we offer alternative hypotheses for the more rapid shedding of fibronectin protein by these cells. 相似文献