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1.
Clin Vaccine Immunol ; 19(10): 1651-60, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22914365

ABSTRACT

On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped prematurely due to documentation issues with the contract manufacturer. Safety and immunogenicity were evaluated through assessments of reactogenicity, reports of adverse events, and assessment of replication-competent and Venezuelan equine encephalitis (VEE) viremia. Immunogenicity was measured using the following assays: enzyme-linked immunosorbent assay (ELISA), chromium 51 ((51)Cr)-release cytotoxic T lymphocyte (CTL), gamma interferon (IFN-γ) ELISpot, intracellular cytokine staining (ICS), and lymphoproliferation assay (LPA). Anti-vector antibodies were also measured. AVX101 was well tolerated and exhibited only modest local reactogenicity. There were 5 serious adverse events reported during the trials; none were considered related to the study vaccine. In contrast to the preclinical data, immune responses in humans were limited. Only low levels of binding antibodies and T-cell responses were seen at the highest doses. This trial also highlighted the difficulties in developing a novel vector for HIV.


Subject(s)
AIDS Vaccines , HIV Antibodies/immunology , HIV Infections/prevention & control , HIV-1/immunology , gag Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/administration & dosage , AIDS Vaccines/adverse effects , AIDS Vaccines/immunology , Adolescent , Adult , Alphavirus/genetics , Botswana , Cytokines/analysis , Double-Blind Method , Encephalomyelitis, Venezuelan Equine/blood , Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunospot Assay , Female , HIV Infections/immunology , HIV-1/classification , HIV-1/genetics , Humans , Interferon-gamma/analysis , Male , Middle Aged , South Africa , T-Lymphocytes, Cytotoxic/immunology , United States , Young Adult
2.
Nat Biotechnol ; 18(12): 1279-82, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11101807

ABSTRACT

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.


Subject(s)
Directed Molecular Evolution/methods , Gene Products, env/genetics , Genetic Vectors , Moloney murine leukemia virus/isolation & purification , Moloney murine leukemia virus/physiology , Recombination, Genetic , Animals , Cell Line , Mice , Moloney murine leukemia virus/genetics , Ultracentrifugation , Virus Replication
3.
Hum Gene Ther ; 10(14): 2337-46, 1999 Sep 20.
Article in English | MEDLINE | ID: mdl-10515453

ABSTRACT

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.


Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins , Fanconi Anemia/therapy , Genetic Therapy , Hematopoietic Stem Cells/metabolism , Nuclear Proteins , Proteins/genetics , Adolescent , Adult , Antigens, CD34/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Child , Colony-Forming Units Assay , Fanconi Anemia/blood , Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group Proteins , Female , Gene Transfer Techniques , Genetic Vectors/genetics , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/pathology , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Male , Retroviridae/genetics
4.
Hum Gene Ther ; 9(17): 2629-40, 1998 Nov 20.
Article in English | MEDLINE | ID: mdl-9853529

ABSTRACT

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.


Subject(s)
Bone Marrow Cells/metabolism , Gaucher Disease/therapy , Genetic Therapy , Glucosylceramidase/genetics , Retroviridae/genetics , Transduction, Genetic , Adult , Antigens, CD34/analysis , Base Sequence , Bone Marrow Cells/immunology , DNA Primers , Female , Gaucher Disease/enzymology , Gaucher Disease/genetics , Gene Transfer Techniques , Genetic Vectors , Glucosylceramidase/blood , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization , Humans , Male , Stromal Cells/cytology
5.
J Virol ; 72(9): 7237-44, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9696818

ABSTRACT

Mouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1(a)). Purified, soluble MHVR expressed from a recombinant vaccinia virus neutralized the infectivity of the A59 strain of mouse hepatitis virus (MHV-A59) in a concentration-dependent manner. Several anchored murine Bgps in addition to MHVR can also function as MHV-A59 receptors when expressed at high levels in nonmurine cells. To investigate the interactions of these alternative MHVR glycoproteins with MHV, we expressed and purified to apparent homogeneity the extracellular domains of several murine Bgps as soluble, six-histidine-tagged glycoproteins, using a baculovirus expression system. These include MHVR isoforms containing four or two extracellular domains and the corresponding Bgp1(b) glycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1(b) comprised of the first two immunoglobulin-like domains. The soluble four-domain MHVR glycoprotein (sMHVR[1-4]) had fourfold more MHV-A59 neutralizing activity than the corresponding soluble Bgp1(b) (sBgp1(b)) glycoprotein and at least 1,000-fold more neutralizing activity than sBgp2. Although virus binds to the N-terminal domain (domain 1), soluble truncation mutants of MHVR and Bgp1(b) containing only domains 1 and 2 bound virus poorly and had 10- and 300-fold less MHV-A59 neutralizing activity than the corresponding four-domain glycoproteins. In contrast, the soluble MHVR glycoprotein containing domains 1 and 4 (sMHVR[1,4]) had as much neutralizing activity as the four-domain glycoprotein, sMHVR[1-4]. Thus, the virus neutralizing activity of MHVR domain 1 appears to be enhanced by domain 4. The sBgp1(b)[1-4] glycoprotein had 500-fold less neutralizing activity for MHV-JHM than for MHV-A59. Thus, MHV strains with differences in S-glycoprotein sequence, tissue tropism, and virulence can differ in the ability to utilize the various murine Bgps as receptors.


Subject(s)
Glycoproteins/immunology , Murine hepatitis virus/metabolism , Receptors, Virus/immunology , 3T3 Cells , Animals , Antigens, CD , Baculoviridae , Cell Adhesion Molecules , Cell Line , Cell Line, Transformed , Chlorocebus aethiops , Genetic Vectors , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Histidine , Mice , Neutralization Tests , Receptors, Virus/isolation & purification , Receptors, Virus/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Solubility , Spodoptera , Vaccinia virus , Vero Cells
6.
Hum Gene Ther ; 7(9): 1095-101, 1996 Jun 10.
Article in English | MEDLINE | ID: mdl-8773511

ABSTRACT

Replication-deficient amphotropic retrovirus vectors (RV) or RV-producer cells are being developed for a variety of human gene therapy strategies. One of the hurdles to in vivo use of these agents is their inactivation by components of human serum. Murine leukemia viruses (MLV), from which most current RV are derived, are known to be inactivated by human serum via activation of the classical complement cascade. Other type C retroviruses, e.g., RD114 and BaEV, are resistant to inactivation by human serum when derived from infection of human and mink cells but not murine cells. We hypothesized that amphotropic RV could be made resistant to human serum inactivation if a more appropriate producer cell could be found. To test this hypothesis, RV were made using a variety of human (293, HOS, TE671) and murine (NIH-3T3) cell types as the producer cell. The parental cell lines, RV-producer cells, and RV themselves were evaluated for sensitivity to inactivation by human serum. Results showed that the murine NIH-3T3 cell line, the NIH-3T3-derived PA317 producer cell line, and RV derived from it were all sensitive to human serum inactivation. In contrast, all human cell lines tested were resistant to lysis. RV and RV-producer cells derived from 293 cells were also resistant; RV derived from HOS cells were resistant. Surprisingly, while TE671 cells were resistant, TE671-derived RV were sensitive to inactivation. To test whether expression of the amphotropic envelope protein was responsible for conferring this serum sensitivity to the RV, env was expressed in the absence of gag and pol in TE671 cells. However, TE671 cells expressing env were resistant to human serum inactivation. These observations have important implications for use of RV and RV-producer cells for human gene therapy.


Subject(s)
Antiviral Agents/physiology , Blood Physiological Phenomena , Gammaretrovirus/genetics , Genetic Therapy/methods , Genetic Vectors , 3T3 Cells , Animals , Cell Line , Humans , Mice , Tumor Cells, Cultured , Virus Replication
7.
Gene Ther ; 2(8): 512-20, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8593601

ABSTRACT

Gaucher disease is a lysosomal storage disorder resulting form deficiency of the acid beta-glucosidase, glucocerebrosidase (GC). Allogeneic bone marrow transplantation has been beneficial in the treatment of Gaucher patients. Therefore, this disorder may be an ideal candidate for gene therapy by GC gene transduction of hematopoietic stem cells. We sought to increase the extent of gene transfer into CD34+ cells from the marrow of a Gaucher patient using G1GC, a simple retroviral vector containing a normal human GC cDNA. The ability of autologous stromal support and recombinant cytokines to increase the extent of transduction of colony-forming-cells (CFCs) and long-term culture initiating cells (LTCICs) was assessed. The presence of a stromal layer significantly increased the extent of GC gene transfer into 14-day CFCs, as determined by polymerase chain reaction (PCR) of individual colonies (18.8% with stroma versus 5% without, P < 0.001). Stromal support also increased the extent of transduction of LTCICs (10% with stroma versus 0.83% without, P < 0.001). Non-adherent cells from long-term bone marrow cultures initiated with CD34+ progenitors transduced on autologous stroma had higher levels of GC enzyme activity than cultures initiated with cells transduced without stroma. The percentage of cells which were GC positive by immunohistochemistry was also increased (21.1% with stroma versus 2.7% without, P = 0.0003). The addition of cytokines (IL-3, IL-6 and Steel factor) to the transduction, in the presence of stroma, significantly increased the extent of gene transfer into CFCs but not LTCICs. These studies indicate that the GC gene can be effectively transduced into LTCICs by retroviral vectors in the presence of stroma at levels significant for clinical gene therapy trials in patients with Gaucher disease.


Subject(s)
Bone Marrow Transplantation/physiology , Gaucher Disease/therapy , Glucosylceramidase/genetics , Hematopoietic Stem Cell Transplantation , Stromal Cells/transplantation , Antigens, CD/analysis , Antigens, CD34/analysis , Bone Marrow Transplantation/pathology , Cells, Cultured , Genetic Therapy/methods , Genetic Vectors , Glucosylceramidase/biosynthesis , Hematopoietic Stem Cells/cytology , Humans , Immunohistochemistry , Polymerase Chain Reaction , Retroviridae , Stromal Cells/pathology , Transformation, Genetic , Transplantation, Autologous
8.
Proc Natl Acad Sci U S A ; 90(5): 1716-20, 1993 Mar 01.
Article in English | MEDLINE | ID: mdl-8383324

ABSTRACT

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.


Subject(s)
Murine hepatitis virus/growth & development , Receptors, Virus/metabolism , Animals , Antibodies, Monoclonal/immunology , Antigens, CD , Base Sequence , Carcinoembryonic Antigen/immunology , Carcinoembryonic Antigen/metabolism , Cell Adhesion Molecules , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , Cricetinae , Glycoproteins/immunology , Glycoproteins/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Murine hepatitis virus/metabolism , Oligodeoxyribonucleotides/chemistry , Protein Processing, Post-Translational , Receptors, Virus/immunology , Recombinant Proteins/metabolism , Sequence Deletion , Structure-Activity Relationship
9.
J Virol ; 67(1): 1-8, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8380065

ABSTRACT

Mouse hepatitis virus-A59 (MHV-A59), a murine coronavirus, can utilize as a cellular receptor MHVR, a murine glycoprotein in the biliary glycoprotein (BGP) subfamily of the carcinoembryonic antigen (CEA) family in the immunoglobulin superfamily (G.S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991). Several different BGP isoforms are expressed in tissues of different mouse strains, and we have explored which of these glycoproteins can serve as functional receptors for MHV-A59. cDNA cloning, RNA-mediated polymerase chain reaction analysis, and Western immunoblotting with a monoclonal antibody, CC1, specific for the N-terminal domain of MHVR showed that the inbred mouse strains BALB/c, C3H, and C57BL/6 expressed transcripts and proteins of the MHVR isoform and/or its splice variants but not the mmCGM2 isoform. In contrast, adult SJL/J mice, which are resistant to infection with MHV-A59, express transcripts and proteins only of the mmCGM2-related isoforms, not MHVR. These data are compatible with the hypothesis that the MHVR and mmCGM2 glycoproteins may be encoded by different alleles of the same gene. We studied binding of anti-MHVR antibodies or MHV-A59 virions to proteins encoded by transcripts of MHVR and mmCGM2 and two splice variants of MHVR, one containing two immunoglobulin-like domains [MHVR(2d)] and the other with four domains as in MHVR but with a longer cytoplasmic domain [MHVR(4d)L]. We found that the three isoforms tested could serve as functional receptors for MHV-A59, although only isoforms that include the N-terminal domain of MHVR were recognized by monoclonal antibody CC1 in immunoblots or by MHV-A59 virions in virus overlay protein blot assays. Thus, in addition to MHVR, both the two-domain isoforms, mmCGM2 and MHVR(2d), and the MHVR(4d)L isoform served as functional virus receptors for MHV-A59. This is the first report of multiple related glycoprotein isoforms that can serve as functional receptors for a single enveloped virus.


Subject(s)
Carcinoembryonic Antigen/metabolism , Glycoproteins/metabolism , Multigene Family , Murine hepatitis virus/metabolism , Receptors, Virus/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Carcinoembryonic Antigen/genetics , Genetic Variation , Glycoproteins/genetics , L Cells , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Murine hepatitis virus/growth & development , RNA Splicing , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Virus/genetics , Recombinant Proteins/genetics , Sequence Homology, Amino Acid
10.
J Virol ; 66(10): 5929-36, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1356160

ABSTRACT

The primary pathophysiologic finding of the viral disease known as Korean hemorrhagic fever, the etiological agent of which is Hantaan virus (HTV), is vascular instability. To investigate whether HTV was able to infect cells derived from human vascular tissue and alter their behavior, we infected in vitro primary adult human endothelial cells from saphenous veins (HSVEC). We were able to detect the presence of viral antigens in infected cells both by immunofluorescence and by Western blot (immunoblot) analysis as early as day 1 postinfection. HSVEC infected with HTV produce infectious virus during the first 3 days of infection but, at later times (days 4 to 8), show decreasing yields of virus. This contrasts with the HTV growth pattern observed for the permissive simian CV-7 cell line, which generates infectious virus up to day 12 after infection. Further investigation showed that the late decrease in viral production in HSVEC is the result of the induction of beta interferon and can be reversed by the addition of anti-beta interferon serum to the culture medium. At no time during the course of infection of HSVEC with HTV was any obvious cytopathic effect observed. When tests for changes in mRNA levels of other cytokines and endothelial cell gene products following HTV infection of HSVEC were done by reverse transcription and polymerase chain reaction methods, no significant changes were observed in the levels of interleukin 1, interleukin 6, or von Willebrand factor mRNA. We hypothesize that, while HTV can replicate in human vascular endothelial cells, the mechanism of microvascular damage seen with Korean hemorrhagic fever is not likely to be a direct effect of virus replication but may conceivably be the consequence of an immune-mediated endothelial injury triggered by viral infection.


Subject(s)
Endothelium, Vascular/microbiology , Orthohantavirus/physiology , Antigens, Viral/analysis , Base Sequence , Blotting, Western , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/immunology , Fluorescent Antibody Technique , Orthohantavirus/isolation & purification , Hemorrhagic Fever with Renal Syndrome/microbiology , Hemorrhagic Fever with Renal Syndrome/physiopathology , Humans , Interleukin-1/genetics , Interleukin-6/genetics , Molecular Sequence Data , Oligonucleotides , Polymerase Chain Reaction , RNA, Messenger/metabolism , Transcription, Genetic , Virus Replication , von Willebrand Factor/genetics
11.
J Virol ; 66(7): 4028-39, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1318394

ABSTRACT

Recently, we showed that a murine member of the carcinoembryonic antigen family of glycoproteins serves as a cellular receptor (MHVR) for the coronavirus mouse hepatitis virus A59 (MHV-A59) (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; R. K. Williams, G.-S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). To examine the role of posttranscriptional modification of MHVR on virus-receptor interactions, a vaccinia virus-based expression system was employed. Expression from the vaccinia virus recombinant (Vac-MHVR) in BHK-21 cells resulted in high levels of MHVR glycoprotein on the cell surface and made these cells susceptible to MHV-A59 infection. Nonglycosylated core MHVR proteins were made in Vac-MHVR-infected BHK-21 cells in the presence of tunicamycin by in vitro translation of MHVR mRNA in a rabbit reticulocyte cell-free system in the absence of microsomal membranes and by expression of an N-terminal deletion clone of MHVR lacking its signal peptide. These three nonglycosylated MHVR proteins were recognized by polyclonal antibody against affinity-purified receptor but did not bind antireceptor monoclonal antibody (MAb) CC1 or MHV-A59 virions. Partial glycosylation of MHVR, either expressed in Vac-MHVR-infected cells treated with monensin or synthesized by in vitro translation with microsomal membranes, restored both the MAb CC1- and the virus-binding activities of the MHVR glycoprotein. Deletion of 26 amino acids at the carboxyl terminus of MHVR resulted in a secreted protein which was able to bind MAb CC1 and MHV-A59. These results suggest that either a carbohydrate moiety is an element of the MHVR-binding site(s) for virus and MAb CC1 or a posttranslational membrane-associated process is required for functional conformation of the receptor glycoprotein.


Subject(s)
Murine hepatitis virus/metabolism , Protein Processing, Post-Translational , Receptors, Virus/metabolism , Vaccinia virus/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA, Viral , Gene Expression , Glycoproteins/metabolism , Immunohistochemistry , Kinetics , Molecular Sequence Data , Murine hepatitis virus/genetics , Receptors, Virus/genetics , Recombinant Proteins/metabolism
12.
J Virol ; 66(4): 1907-14, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1548747

ABSTRACT

The two glycoproteins of Hantaan virus (HTV), G1 and G2, are encoded as a continuous single open reading frame in the M segment of the virion RNA. They are believed to be synthesized contemporaneously via a polypeptide precursor which is then processed to yield two glycoproteins, both of which appear in the Golgi complex of the cell. To study the properties of G1 and G2 as separate entities, we have constructed vaccinia virus recombinants which contain the sequences for each glycoprotein individually. Both glycoproteins made from these recombinants appear normal on sodium dodecyl sulfate-polyacrylamide gels compared with HTV products made in virus-infected cells. Interestingly, in the independently expressed G2 recombinant, a stretch of hydrophobic amino acids preceding the mature G2 N terminus appears to contain the signals necessary for translocation across membranes and proper glycosylation; partial deletion of this hydrophobic sequence results in production of an nonglycosylated form of G2. Thus, both G1 and G2 appear able to be expressed in an authentic fashion quite independently of each other, using their own signal sequences. In addition, it appears that the G1 from vaccinia virus recombinants contains the motif(s) necessary for cellular targeting of the HTV glycoproteins, while G2 from vaccinia virus recombinants remains strongly associated with the endoplasmic reticulum. In contrast, cells doubly infected with G1-vaccinia virus and G2-vaccinia virus recombinants show the G2 in a predominantly perinuclear (Golgi-like) distribution, presumably targeted there through association with G1. A carboxy-terminal deletion of G1 (2-43-Vac), which lacks 82 amino acids proximal to the start of the mature G2, retains a Golgi-like distribution.


Subject(s)
Gene Expression Regulation, Viral , Glycoproteins/genetics , Orthohantavirus/genetics , Viral Proteins/genetics , Biological Transport , Blotting, Western , Cell Line , Cloning, Molecular , Fluorescent Antibody Technique , Gene Expression Regulation, Viral/drug effects , Glycoproteins/metabolism , Golgi Apparatus/metabolism , Open Reading Frames , Protein Processing, Post-Translational , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tunicamycin/pharmacology , Vaccinia virus/genetics , Viral Proteins/metabolism
13.
J Virol ; 65(12): 6881-91, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1719235

ABSTRACT

The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59.


Subject(s)
Genes, Immunoglobulin , Multigene Family , Murine hepatitis virus/physiology , Receptors, Virus/physiology , Transfection , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cell Line , Cloning, Molecular , Colon/microbiology , Colon/physiology , Cricetinae , Fluorescent Antibody Technique , Genetic Predisposition to Disease , Humans , Mice , Mice, Inbred Strains , Molecular Sequence Data , Murine hepatitis virus/pathogenicity , Oligodeoxyribonucleotides , Polymerase Chain Reaction/methods , Protein Conformation , RNA/genetics , RNA/isolation & purification , Receptors, Virus/genetics , Virus Replication
14.
J Virol ; 64(12): 6101-9, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2173782

ABSTRACT

The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-1-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a gamma 1 gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCl gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication.


Subject(s)
Genes, Viral , Open Reading Frames , Simplexvirus/genetics , Viral Proteins/genetics , Animals , Cell Line , DNA, Viral/genetics , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/isolation & purification , Molecular Weight , Plasmids , Protein Biosynthesis , Restriction Mapping , Sequence Homology, Nucleic Acid , Simplexvirus/enzymology , Transcription, Genetic , Vero Cells , Viral Proteins/isolation & purification
15.
J Virol ; 64(9): 4540-8, 1990 Sep.
Article in English | MEDLINE | ID: mdl-2166829

ABSTRACT

Many characteristics of the putative protein encoded by varicella-zoster virus (VZV) open reading fram (ORF) 14 indicate that it is a glycoprotein, which has been designated gpV. To identify the protein products of the gene, the coding sequences were placed under the control of the vaccinia virus p7.5 promoter and recombinant vaccinia viruses were constructed. Heterogeneous polypeptides with molecular weights of 95,000 to 105,000 (95K to 105K polypeptides) were expressed in cells infected by a vaccinia virus recombinant (vKIP5) containing ORF 14 from VZV Scott but were not expressed by control vaccinia viruses. These polypeptides were recognized by antibodies present in human sera that contained high levels of anti-VZV antibodies. Conversely, antisera raised in rabbits inoculated with vKIP5 reacted specifically with heterogeneous 95K to 105K polypeptides present in VZV Scott-infected but not uninfected cells; these polypeptides show a patchy plasma membrane fluorescence pattern in VZV Scott-infected cells. These same antisera neutralized VZV strain Scott infectivity in the absence of complement. Endoglycosidase F treatment of isolated gpV polypeptides and tunicamycin treatment of cells infected with the vKIP5 recombinant indicated that the polypeptides were glycosylated. Three sets of data imply that the VZV strain Oka, which has been used to produce a live attenuated virus vaccine, accumulates low levels of gpV polypeptides relative to wild-type strains: (i) blocking of antibodies in human sera with excess VZV Oka-infected cell antigen yielded residual antibodies which were reactive with the 95K to 105K gpV polypeptides expressed in cells infected by VZV strain Scott and by the vKIP5 vaccinia virus recombinant, but not with Oka-infected cell polypeptides; (ii) antisera raised to vKIP5 detected very low levels of reactive polypeptides made in VZV Oka-infected cells and neutralized VZV Oka virus much less efficiently than VZV Scott; and (iii) comparisons of the reactivity of sera from live attenuated virus vaccine vaccinees with sera derived from patients recovering from wild-type infections indicated greatly reduced levels of gpV-specific antibodies in some vaccinees.


Subject(s)
Genes, Viral , Glycoproteins/genetics , Herpesvirus 3, Human/genetics , Viral Proteins/genetics , Animals , Base Sequence , Cell Line , Cloning, Molecular , DNA, Viral/genetics , DNA, Viral/isolation & purification , Fluorescent Antibody Technique , Humans , Immune Sera , Molecular Sequence Data , Neutralization Tests , Plasmids , Promoter Regions, Genetic , Restriction Mapping , Vaccinia virus/genetics
17.
J Virol ; 62(3): 696-702, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3123711

ABSTRACT

A cDNA containing the complete open reading frame of the Hantaan virus (HTN) M genome segment has been cloned into vaccinia virus. This recombinant virus expresses two glycoproteins which are similar to the HTN structural glycoproteins, G1 and G2, in molecular weight, cleavage pattern, and cellular distribution. Both HTN and recombinant vaccinia virus glycoproteins are exclusively associated with the Golgi apparatus of the cell. Despite this intracellular restriction, mice inoculated with the recombinant vaccinia virus raised neutralizing antibodies against HTN. The specificity of virus neutralization appears to reside in the HTN glycoproteins, since a vaccinia virus recombinant expressing the HTN nucleocapsid protein was unable to elicit a neutralizing antibody response.


Subject(s)
Genes, Viral , Orthohantavirus/genetics , Animals , Antibodies, Viral/biosynthesis , DNA/genetics , Gene Expression Regulation , Glycoproteins/genetics , Glycoproteins/immunology , Orthohantavirus/immunology , Mice , Mice, Inbred BALB C/immunology , Vaccinia virus/genetics , Vaccinia virus/immunology , Viral Proteins/genetics , Viral Proteins/immunology , Viral Structural Proteins
18.
J Gerontol Nurs ; 13(10): 11-7, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3668176
19.
J Virol ; 56(1): 161-71, 1985 Oct.
Article in English | MEDLINE | ID: mdl-2993647

ABSTRACT

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.


Subject(s)
Enterovirus Infections/genetics , Gene Expression Regulation , Mengovirus/genetics , Protein Biosynthesis , Ribosomes/physiology , Viral Proteins/isolation & purification , Animals , Kinetics , L Cells , Mice , Polyribosomes/physiology , Pronase , Time Factors
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