Marie-Louise Hammarskjold | University of Virginia (original) (raw)

Papers by Marie-Louise Hammarskjold

Research paper thumbnail of High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

Gene, 1986

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV... more To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Research paper thumbnail of Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP)

Immunology Letters, Aug 1, 1988

Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detecte... more Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji ceils and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBVseropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Research paper thumbnail of Intron Retention Generates a Novel Id3 Isoform That Inhibits Vascular Lesion Formation

Journal of Biological Chemistry, Jul 1, 2004

Research paper thumbnail of Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum

Journal of Virological Methods, Jul 1, 1986

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate Ba... more We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamHl-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Barn K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Barn K containing vector, the apparent molecular weights of the 4 major bands are 74,68,62 and 57 kD. Labelling of transfected cells with ['Hlglycine and ['*P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes. Epstein-Barr virus immunoprecipitation human serum EBNAl

Research paper thumbnail of Expression of Ebv Ebna Proteins Using an SV 40 Late Replacement Vector

Research paper thumbnail of Intra-arterial delivery of adenovirus stimulates neointimal formation and negates the benefit of adenovirus-hirudin in a rabbit arterial injury model

Research paper thumbnail of Mutations in the principal neutralization domain of the human immunodeficiency virus type 1 affect syncytium formation, virus infectivity, growth kinetics, and neutralization

Research paper thumbnail of Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells

Nature, 1987

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The ... more The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.

Research paper thumbnail of Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum

Journal of Virological Methods, 1986

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate Ba... more We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamHl-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Barn K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Barn K containing vector, the apparent molecular weights of the 4 major bands are 74,68,62 and 57 kD. Labelling of transfected cells with ['Hlglycine and ['*P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes. Epstein-Barr virus immunoprecipitation human serum EBNAl

Research paper thumbnail of Intron Retention Generates a Novel Id3 Isoform That Inhibits Vascular Lesion Formation

Journal of Biological Chemistry, 2004

Research paper thumbnail of Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP)

Immunology Letters, 1988

Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detecte... more Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji ceils and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBVseropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Research paper thumbnail of High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

Gene, 1986

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV... more To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Research paper thumbnail of Intron retention in viruses and cellular genes: Detention, border controls and passports

WIREs RNA, 2018

Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thoug... more Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thought to be rare in mammalian cells, albeit common in plants and some viruses. Largely due to the development of better methods for RNA analysis, it has now been recognized that IR is much more common than previously thought and that this mechanism is likely to play an important role in mammalian gene regulation. To date, most publications and reviews about IR have described the resulting mRNAs as “dead end” products, with no direct consequence for the proteome. However, there are also many reports of mRNAs with retained introns giving rise to alternative protein isoforms. Although this was originally revealed in viral systems, there are now numerous examples of bona fide cellular proteins that are translated from mRNAs with retained introns. These new isoforms have sometimes been shown to have important regulatory functions. In this review, we highlight recent developments in this area and...

Research paper thumbnail of HIV-1 Rev-RRE Functional Activity in Primary Isolates is Highly Dependent on Minimal Context-Dependent Changes in Rev

During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host... more During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host cell nucleus to the cytoplasm in order to complete the replication cycle. To overcome cellular restrictions to export incompletely spliced transcripts, HIV encodes a protein, Rev, that is constitutively expressed from a completely spliced transcript. Rev is then imported into the nucleus where it binds to an RNA structure on intron-containing viral mRNAs called the Rev Response Element (RRE). Bound Rev multimerizes and recruits cellular factors that permit the nuclear export of the resulting ribonucleoprotein complex. Primary HIV isolates display substantial variation in the functional activity of the Rev-RRE axis, which may permit viral adaptation to differing immune environments. We describe two subtype G primary isolates with disparate Rev activity. Rev activity was correlated within vitrofitness of replication-competent viral constructs. Amino acid differences within the oligomerzia...

Research paper thumbnail of Rev-Rev Response Element Activity Selection Bias at the HIV Transmission Bottleneck

HIV is not efficiently transmitted between hosts, and selection of viral variants occurs during t... more HIV is not efficiently transmitted between hosts, and selection of viral variants occurs during the process of sexual transmission. The factors that confer selective advantage at the transmission bottleneck remain incompletely understood. We explored whether differences in the Rev-Rev Response Element (RRE) regulatory axis of HIV affect transmission fitness, since functional variation in the Rev-RRE axis in different viral isolates has been shown to affect replication kinetics and relative expression of many HIV proteins. Single genome HIV sequences were identified from nine linked subject pairs near the time of female-to-male transmission. Using a rapid flow-cytometric assay, we found that the functional Rev-RRE activity varied significantly between isolates. Moreover, it was generally lower in recipients’ viruses compared to the corresponding donor viruses. In six of nine transmission events, recipient virus Rev-RRE activity clustered at the extreme low end of the range of donor v...

Research paper thumbnail of Altered-Self MHC Class I Sensing via Functionally Disparate Paired NK Cell Receptors Counters Murine Cytomegalovirus gp34–Mediated Immune Evasion

The Journal of Immunology

The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface ... more The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface of infected cells where these complexes bind Ly49 inhibitory receptors (IRs) and prevent NK cell attack. Nonetheless, certain self–MHC I–binding Ly49 activating and inhibitory receptors are able to promote robust NK cell expansion and antiviral immunity during MCMV infection. A basis for MHC I-dependent NK cell sensing of MCMV-infected targets and control of MCMV infection however remains unclear. In this study, we discovered that the Ly49R activation receptor is selectively triggered during MCMV infection on antiviral NK cells licensed by the Ly49G2 IR. Ly49R activating receptor recognition of MCMV-infected targets is dependent on MHC I Dk and MCMV gp34 expression. Remarkably, although Ly49R is critical for Ly49G2-dependent antiviral immunity, blockade of the activation receptor in Ly49G2-deficient mice has no impact on virus control, suggesting that paired Ly49G2 MCMV sensing might ena...

Research paper thumbnail of The Replication Activity of SV40 DNA Correlates with the Level of c-myc Expression in Human Tumor Cell Lines

Current Topics in Microbiology and Immunology, 1988

Research paper thumbnail of Human endogenous retrovirus-K mRNA expression and genomic alignment data in hepatoblastoma

Research paper thumbnail of Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase

Journal of Ethnopharmacology, 2020

Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunode... more Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances. Aim of the study: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity. Materials and methods: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4 + /CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC 50 values in the nanomolar range. Results: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 μM (IC 50 : 0.49 μM, CC 50 : 3089 μM) and 1343 μM (IC 50 : 2.3 μM, CC 50 : 3089 μM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC 50 values of 11.11 μg/ml and 16.86 μg/ml, respectively. Conclusion: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.

Research paper thumbnail of Upregulation of Human Endogenous Retrovirus-K (HML-2) mRNAs in hepatoblastoma: Identification of potential new immunotherapeutic targets and biomarkers

PurposeHepatoblastoma is the most common liver malignancy in children. In order to advance therap... more PurposeHepatoblastoma is the most common liver malignancy in children. In order to advance therapy against hepatoblastoma, novel immunologic targets and biomarkers are needed. Our purpose in this investigation is to examine hepatoblastoma transcriptomes for the expression of a class of genomic elements known as Human Endogenous Retrovirus (HERVs). HERVs are abundant in the human genome and are biologically active elements that have been associated with multiple malignancies and proposed as immunologic targets in a subset of tumors. A sub-family of HERVs, HERV-K (HML-2), have been shown to be tightly regulated in fetal development, making investigation of these elements in pediatric tumors paramount.MethodsWe first created a HERVK-FASTA file utilizing 91 previously described HML-2 proviruses. We then concatenated the file onto the GRCh38.95 cDNA library from Ensembl. We used this computational tool to evaluate existing RNA-seq data from 10 hepatoblastoma tumors and 3 normal liver con...

Research paper thumbnail of High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

Gene, 1986

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV... more To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Research paper thumbnail of Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP)

Immunology Letters, Aug 1, 1988

Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detecte... more Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji ceils and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBVseropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Research paper thumbnail of Intron Retention Generates a Novel Id3 Isoform That Inhibits Vascular Lesion Formation

Journal of Biological Chemistry, Jul 1, 2004

Research paper thumbnail of Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum

Journal of Virological Methods, Jul 1, 1986

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate Ba... more We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamHl-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Barn K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Barn K containing vector, the apparent molecular weights of the 4 major bands are 74,68,62 and 57 kD. Labelling of transfected cells with ['Hlglycine and ['*P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes. Epstein-Barr virus immunoprecipitation human serum EBNAl

Research paper thumbnail of Expression of Ebv Ebna Proteins Using an SV 40 Late Replacement Vector

Research paper thumbnail of Intra-arterial delivery of adenovirus stimulates neointimal formation and negates the benefit of adenovirus-hirudin in a rabbit arterial injury model

Research paper thumbnail of Mutations in the principal neutralization domain of the human immunodeficiency virus type 1 affect syncytium formation, virus infectivity, growth kinetics, and neutralization

Research paper thumbnail of Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells

Nature, 1987

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The ... more The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.

Research paper thumbnail of Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum

Journal of Virological Methods, 1986

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate Ba... more We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamHl-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Barn K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Barn K containing vector, the apparent molecular weights of the 4 major bands are 74,68,62 and 57 kD. Labelling of transfected cells with ['Hlglycine and ['*P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes. Epstein-Barr virus immunoprecipitation human serum EBNAl

Research paper thumbnail of Intron Retention Generates a Novel Id3 Isoform That Inhibits Vascular Lesion Formation

Journal of Biological Chemistry, 2004

Research paper thumbnail of Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP)

Immunology Letters, 1988

Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detecte... more Antibodies reactive with the Epstein-Barr (EBV)encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji ceils and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBVseropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Research paper thumbnail of High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

Gene, 1986

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV... more To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Research paper thumbnail of Intron retention in viruses and cellular genes: Detention, border controls and passports

WIREs RNA, 2018

Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thoug... more Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thought to be rare in mammalian cells, albeit common in plants and some viruses. Largely due to the development of better methods for RNA analysis, it has now been recognized that IR is much more common than previously thought and that this mechanism is likely to play an important role in mammalian gene regulation. To date, most publications and reviews about IR have described the resulting mRNAs as “dead end” products, with no direct consequence for the proteome. However, there are also many reports of mRNAs with retained introns giving rise to alternative protein isoforms. Although this was originally revealed in viral systems, there are now numerous examples of bona fide cellular proteins that are translated from mRNAs with retained introns. These new isoforms have sometimes been shown to have important regulatory functions. In this review, we highlight recent developments in this area and...

Research paper thumbnail of HIV-1 Rev-RRE Functional Activity in Primary Isolates is Highly Dependent on Minimal Context-Dependent Changes in Rev

During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host... more During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host cell nucleus to the cytoplasm in order to complete the replication cycle. To overcome cellular restrictions to export incompletely spliced transcripts, HIV encodes a protein, Rev, that is constitutively expressed from a completely spliced transcript. Rev is then imported into the nucleus where it binds to an RNA structure on intron-containing viral mRNAs called the Rev Response Element (RRE). Bound Rev multimerizes and recruits cellular factors that permit the nuclear export of the resulting ribonucleoprotein complex. Primary HIV isolates display substantial variation in the functional activity of the Rev-RRE axis, which may permit viral adaptation to differing immune environments. We describe two subtype G primary isolates with disparate Rev activity. Rev activity was correlated within vitrofitness of replication-competent viral constructs. Amino acid differences within the oligomerzia...

Research paper thumbnail of Rev-Rev Response Element Activity Selection Bias at the HIV Transmission Bottleneck

HIV is not efficiently transmitted between hosts, and selection of viral variants occurs during t... more HIV is not efficiently transmitted between hosts, and selection of viral variants occurs during the process of sexual transmission. The factors that confer selective advantage at the transmission bottleneck remain incompletely understood. We explored whether differences in the Rev-Rev Response Element (RRE) regulatory axis of HIV affect transmission fitness, since functional variation in the Rev-RRE axis in different viral isolates has been shown to affect replication kinetics and relative expression of many HIV proteins. Single genome HIV sequences were identified from nine linked subject pairs near the time of female-to-male transmission. Using a rapid flow-cytometric assay, we found that the functional Rev-RRE activity varied significantly between isolates. Moreover, it was generally lower in recipients’ viruses compared to the corresponding donor viruses. In six of nine transmission events, recipient virus Rev-RRE activity clustered at the extreme low end of the range of donor v...

Research paper thumbnail of Altered-Self MHC Class I Sensing via Functionally Disparate Paired NK Cell Receptors Counters Murine Cytomegalovirus gp34–Mediated Immune Evasion

The Journal of Immunology

The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface ... more The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface of infected cells where these complexes bind Ly49 inhibitory receptors (IRs) and prevent NK cell attack. Nonetheless, certain self–MHC I–binding Ly49 activating and inhibitory receptors are able to promote robust NK cell expansion and antiviral immunity during MCMV infection. A basis for MHC I-dependent NK cell sensing of MCMV-infected targets and control of MCMV infection however remains unclear. In this study, we discovered that the Ly49R activation receptor is selectively triggered during MCMV infection on antiviral NK cells licensed by the Ly49G2 IR. Ly49R activating receptor recognition of MCMV-infected targets is dependent on MHC I Dk and MCMV gp34 expression. Remarkably, although Ly49R is critical for Ly49G2-dependent antiviral immunity, blockade of the activation receptor in Ly49G2-deficient mice has no impact on virus control, suggesting that paired Ly49G2 MCMV sensing might ena...

Research paper thumbnail of The Replication Activity of SV40 DNA Correlates with the Level of c-myc Expression in Human Tumor Cell Lines

Current Topics in Microbiology and Immunology, 1988

Research paper thumbnail of Human endogenous retrovirus-K mRNA expression and genomic alignment data in hepatoblastoma

Research paper thumbnail of Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase

Journal of Ethnopharmacology, 2020

Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunode... more Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances. Aim of the study: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity. Materials and methods: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4 + /CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC 50 values in the nanomolar range. Results: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 μM (IC 50 : 0.49 μM, CC 50 : 3089 μM) and 1343 μM (IC 50 : 2.3 μM, CC 50 : 3089 μM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC 50 values of 11.11 μg/ml and 16.86 μg/ml, respectively. Conclusion: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.

Research paper thumbnail of Upregulation of Human Endogenous Retrovirus-K (HML-2) mRNAs in hepatoblastoma: Identification of potential new immunotherapeutic targets and biomarkers

PurposeHepatoblastoma is the most common liver malignancy in children. In order to advance therap... more PurposeHepatoblastoma is the most common liver malignancy in children. In order to advance therapy against hepatoblastoma, novel immunologic targets and biomarkers are needed. Our purpose in this investigation is to examine hepatoblastoma transcriptomes for the expression of a class of genomic elements known as Human Endogenous Retrovirus (HERVs). HERVs are abundant in the human genome and are biologically active elements that have been associated with multiple malignancies and proposed as immunologic targets in a subset of tumors. A sub-family of HERVs, HERV-K (HML-2), have been shown to be tightly regulated in fetal development, making investigation of these elements in pediatric tumors paramount.MethodsWe first created a HERVK-FASTA file utilizing 91 previously described HML-2 proviruses. We then concatenated the file onto the GRCh38.95 cDNA library from Ensembl. We used this computational tool to evaluate existing RNA-seq data from 10 hepatoblastoma tumors and 3 normal liver con...