Antigenicity and hemaglutination activity of a recombinant Hemagglutinin-neuraminidase of paramyxovirus Tianjin strain

Paramyxovirus Tianjin strain, a new genotype of Sendal virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity.The segment rHN2 possesses more linear epitopes exposed on the surface of the native I-IN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinicalserum specimens.     

55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.     

Identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein p15E required for inhibition of lymphoproliferation and its similarity to gp21 of human T-lymphotropic virus types I and II.

Synthetic peptides containing portions of a highly conserved region of retroviral transmembrane proteins of human and animal retroviruses were tested for their ability to inhibit lymphoproliferation to determine the minimum amino acid sequence required. The previously reported immunosuppression mediated by the peptide CKS-17 was confirmed and further localized to a sequence of eight residues essentially identical to the sequence present in the transmembrane protein gp21 of human T-lymphotropic virus types I and II (HTLV-I and -II). To substantiate the physiological relevance of the inhibition of lymphoproliferation observed with the synthetic peptides and to relate this activity to the intact protein, we purified the Rauscher murine leukemia virus transmembrane protein p15E by immunoaffinity chromatography and report that this purified component presented in the form of protein micelles inhibited the interleukin-2-dependent proliferation of the murine T-cell line CTLL-2 in a dose-dependent manner, with a half-maximal inhibitory dose (ID50) of approximately 16 nM. In comparison, the ID50 concentration of a recombinant form of p15E required to inhibit lymphoproliferation was approximately 2.2 microM. The results reported here support the hypothesis that the transmembrane protein gp21 of HTLV-I and -II participates in the mechanism of immunosuppression previously reported for the transmembrane proteins of feline leukemia virus and other animal retroviruses. Thus, the transmembrane protein of HTLV-I, the etiological agent of adult T-cell leukemia-lymphoma, may be partially responsible for the immunocompromised clinical course of this disease that results in fatal opportunistic infections in a majority of cases.     

Role of conserved gp41 cysteine residues in the processing of human immunodeficiency virus envelope precursor and viral infectivity.

All animal retroviruses whose nucleotide sequences have been determined contain two or three closely spaced cysteine residues in the extracellular domain of the env-encoded transmembrane protein. Using human immunodeficiency virus type 1 gp41 as a working model, the functional significance of these highly conserved cysteines was investigated. We report here that substituting the two conserved cysteine residues in this domain of gp41 with glycine residues resulted in the loss of viral infectivity, which could be attributed to severe impairment in the processing of gp160 precursor to gp120.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.     

Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector.

In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

Inhibition of lymphoproliferation by a synthetic peptide with sequence identity to gp41 of human immunodeficiency virus type 1.

Peptides were synthesized that contained sequences from two regions (env amino acids [aa] 581 to 597 and 655 to 671) of the transmembrane protein gp41 and one region of the external envelope glycoprotein gp120 (aa 457 to 464) of human immunodeficiency virus type 1. Selection of these sequences was based on their homology to the highly conserved and immunosuppressive sequence contained within the transmembrane proteins p15E and gp21 of animal and human retroviruses, respectively. Peptide aa581-597 was found to specifically inhibit human and murine lymphoproliferation, whereas peptides aa655-671 and aa457-464 had no activity. These results suggest a mechanism by which human immunodeficiency virus type 1 gp41 exerts a direct immunosuppressive effect in vivo, analogous to that postulated for p15E and gp21, which could contribute to the immune dysfunction observed in patients suffering from acquired immunodeficiency syndrome. It is of particular interest that the sequence aa 584 to 609, shown to contain B- and T-helper-cell epitopes, overlaps with the sequence aa 581 to 597 that is shown here to inhibit lymphoproliferation. The potential implications of this overlap of immunologic activities are discussed.     

Envelope proteins of human T cell leukemia virus type I: characterization by antisera to synthetic peptides and identification of a natural epitope

Three peptides corresponding to selected regions of the env gene products of human T cell leukemia virus type I were synthesized by solid-phase Merrifield techniques. The sequence of peptide designated SP-65 was identical to the predicted C-terminal 12 residues of the transmembrane protein p21env, and peptide SP-74 was inferred from a region shown to be highly conserved among mammalian retroviruses. The third peptide, SP-70, was derived from a C-terminal region of the surface glycoprotein gp46. Antibodies to each peptide were raised in rabbits and were used to identify and further characterize the proteins coded by the env gene. Despite being present at very low levels in purified viral preparations, these proteins were chromatographed by reverse-phase high pressure liquid chromatography and were located by Western blot analysis of the column fractions. Anti-SP-70 recognized the surface glycoprotein (gp46) and also its C-terminal cleavage fragment (gp16). Anti-SP-65 and anti-SP-74 both reacted with the hydrophobic transmembrane protein (p21) and provided evidence that this protein does not undergo apparent C-terminal processing during viral maturation, unlike the trans-membrane protein of murine leukemia virus. As expected, anti-SP-74 also reacted with homologous proteins from other Type C and Type D viruses, confirming that peptide SP-74 corresponds to a broadly conserved region of retroviral transmembrane proteins. SP-70, which is predicted to be quite near the C terminus of the major surface glycoprotein, was also reactive with sera of HTLV-I-positive patients, indicating that this peptide corresponds to, or is part of, a native epitope recognized by the natural host.     

Major Group-Specific Protein of Rat Type C Viruses

The major internal protein of a rat type C virus pseudotype of murine sarcoma virus, MSV(RaLV), was purified by isoelectric focusing (pI = 8.6) and used to prepare antibody in guinea pigs. The protein was identified by its reaction with antisera reactive with the mammalian type C virus group-specific (gs) antigenic determinant, gs-3. The guinea pig antisera mainly contained species-specific (gs-1) antibody for reactions in gel diffusion with other type C viruses were limited to those of rat origin, whereas in complement fixation tests heterologous reactions could be eliminated by use of appropriate antiserum concentrations without affecting homologous reactions. Guinea pig antisera against mouse, hamster, or cat gs-1 determinants did not react with MSV(RaLV) purified gs protein or with any of several other rat type C viruses.     

Sodium-dependent neutral amino acid transporter type 1 is an auxiliary receptor for baboon endogenous retrovirus

The baboon endogenous retrovirus (BaEV) belongs to a large, widely dispersed interference group that includes the RD114 feline endogenous virus and primate type D retroviruses. Recently, we and another laboratory independently cloned a human receptor for these viruses and identified it as the human sodium-dependent neutral amino acid transporter type 2 (hASCT2). Interestingly, mouse and rat cells are efficiently infected by BaEV but only become susceptible to RD114 and type D retroviruses if the cells are pretreated with tunicamycin, an inhibitor of protein N-linked glycosylation. To investigate this host range difference, we cloned and analyzed NIH Swiss mouse ASCT2 (mASCT2). Surprisingly, mASCT2 did not mediate BaEV infection, which implied that mouse cells might have an alternative receptor for this virus. In addition, elimination of the two N-linked oligosaccharides from mASCT2 by mutagenesis, as substantiated by protein N-glycosidase F digestions and Western immunoblotting, did not enable it to function as a receptor for RD114 or type D retroviruses. Based on these results, we found that the related ASCT1 transporters of humans and mice are efficient receptors for BaEV but are relatively inactive for RD114 and type D retroviruses. Furthermore, elimination of the two N-linked oligosaccharides from extracellular loop 2 of mASCT1 by mutagenesis enabled it to function as an efficient receptor for RD114 and type D retroviruses. Thus, we infer that the tunicamycin-dependent infection of mouse cells by RD114 and type D retroviruses is caused by deglycosylation of mASCT1, which unmasks previously buried sites for viral interactions. In contrast, BaEV efficiently employs the glycosylated forms of mASCT1 that occur normally in untreated mouse cells.     

The three-dimensional solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer monkey virus, and implications for the morphology of retroviral assembly.

The Mason-Pfizer monkey virus (M-PMV) is the prototype of the type D retroviruses. In type B and D retroviruses, the Gag protein pre-assembles before association with the membrane, whereas in type C retroviruses (lentiviruses, BLV/HTLV group) Gag is targeted efficiently to the plasma membrane, where the particle formation occurs. The N-terminal domain of Gag, the matrix protein (MA), plays a critical role in determining this morphogenic difference. We have determined the three-dimensional solution structure of the M-PMV MA by heteronuclear nuclear magnetic resonance. The protein contains four alpha-helices that are structurally similar to the known type C MA structures. This similarity implies possible common assembly units and membrane-binding mechanisms for type C and B/D retroviruses. In addition to this, the interpretation of mutagenesis data has enabled us to identify, for the first time, the structural basis of a putative intracellular targeting motif.     

Spleen necrosis virus, an avian immunosuppressive retrovirus, shares a receptor with the type D simian retroviruses.

The reticuloendotheliosis viruses (REV) are a family of highly related retroviruses isolated from gallinaceous birds. On the basis of sequence comparison and overall genome organization, these viruses are more similar to the mammalian type C retroviruses than to the avian sarcoma/leukemia viruses. The envelope of a member of the REV family, spleen necrosis virus (SNV), is about 50% identical in amino acid sequence to the envelope of the type D simian retroviruses. Although SNV does not productively infect primate or murine cells, the receptor for SNV is present on a variety of human and murine cells. Moreover, interference assays show that the receptor for SNV is the same as the receptor for the type D simian retroviruses. We propose that adaptation of a mammalian type C virus to an avian host provided the REV progenitor.     

In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid.

It has recently been shown by mass spectral analysis (Henderson et al., Proc. Natl. Acad. Sci. U.S.A. 80:339-343, 1983) that the p15gag protein of murine leukemia viruses contains a novel post-translational modification, an amino-terminal myristyl (tetradecanoyl) amide. In this report we show that p15gag is the only structural protein to contain this fatty acid. In addition, the gag precursor polyproteins of type B, C, and D retroviruses have been examined for the presence of myristic acid by metabolic labeling and immunoprecipitation studies. In a panel of mammalian type C retroviruses we found that the precursor polyprotein Pr65gag homologs, but not the glycosylated forms (gPr80gag homologs), were specifically labeled after a 5-min incubation of infected cells with [3H]myristic acid. The gag precursor polyprotein was also labeled in mouse mammary tumor virus and in Mason-Pfizer monkey virus, but Pr76gag of Rous sarcoma virus failed to incorporate [3H]myristate. Under similar conditions, [3H]palmitate was not found to be incorporated into any viral gag proteins. Thus, myristylation appears to be a common feature of mammalian type B, C, and D retroviruses but not of avian retroviruses.     

Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity.     

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333–340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.     

Role of gag sequence in the biochemical properties and transforming activity of the avian sarcoma virus UR2-encoded gag-ros fusion protein.

The transforming protein P68gag-ros of avian sarcoma virus UR2 is a transmembrane tyrosine protein kinase molecule with the gag portion protruding extracellularly. To investigate the role of the gag moiety in the biochemical properties and biological functions of the P68gag-ros fusion protein, retroviruses containing the ros coding sequence of UR2 were constructed and analyzed. The gag-free ros protein was expressed from one of the mutant retroviruses at a level 10 to 50% of that of the wild-type UR2. However, the gag-free ros-containing viruses were not able to either transform chicken embryo fibroblasts or induce tumors in chickens. The specific tyrosine protein kinase activity of gag-free ros protein is about 10- to 20-fold reduced as judged by in vitro autophosphorylation. The gag-free ros protein is still capable of associating with membrane fractions including the plasma membrane, indicating that sequences essential for recognition and binding membranes must be located within ros. Upon passages of the gag-free mutants, transforming and tumorigenic variants occasionally emerged. The variants were found to have regained the gag sequence fused to the 5' end of the ros, apparently via recombination with the helper virus or through intramolecular recombination between ros and upstream gag sequences in the same virus construct. All three variants analyzed code for gag-ros fusion protein larger than 68 kDa. The gag-ros recombination junction of one of the transforming variants was sequenced and found to consist of a p19-p10-p27-ros fusion sequence. We conclude that the gag sequence is essential for the transforming activity of P68gag-ros but is not important for its membrane association.     

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells.

The influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. By using deletion mutants and chimeric constructs of influenza virus NA with the human transferrin receptor, a type II basolateral transmembrane protein, we investigated the location of the apical sorting signal of influenza virus NA. When these mutant and chimeric proteins were expressed in stably transfected polarized MDCK cells, the transmembrane domain of NA, and not the cytoplasmic tail, provided a determinant for apical targeting in polarized MDCK cells and this transmembrane signal was sufficient for sorting and transport of the ectodomain of a reporter protein (transferrin receptor) directly to the apical plasma membrane of polarized MDCK cells. In addition, by using differential detergent extraction, we demonstrated that influenza virus NA and the chimeras which were transported to the apical plasma membrane also became insoluble in Triton X-100 but soluble in octylglucoside after extraction from MDCK cells during exocytic transport. These data indicate that the transmembrane domain of NA provides the determinant(s) both for apical transport and for association with Triton X-100-insoluble lipids.