Marie-Louise Hammarskjold - Profile on Academia.edu (original) (raw)

Papers by Marie-Louise Hammarskjold

Journal of Pediatric Surgery, Feb 1, 2021

Purpose: Hepatoblastoma is the most common liver malignancy in children. In order to advance ther... more Purpose: Hepatoblastoma 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. We 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 controls (GEO accession ID: GSE89775). Quantification and differential proviral expression analysis between hepatoblastoma and normal liver controls was performed using the pseudo-alignment program Salmon and DESeq2, respectively. Results: HERV-K mRNA was expressed in hepatoblastoma from multiple proviral loci. All HERV-K proviral loci were expressed at higher levels in hepatoblastoma compared to normal liver controls. Five 3q27.2, 7q22.2, 12q24.33 and 17p13.1) were significantly differentially expressed (p-adjusted value < 0.05, |log2 fold change| > 1.5) across conditions. The provirus at 17p13.1 had an approximately 300-fold increased expression in hepatoblastoma as compared to normal liver. This was in part due to the near absence of HERV-K mRNA at the 17p13.1 locus in fully differentiated liver samples. Conclusions: Our investigation demonstrates that HERV-K is expressed from multiple loci in hepatoblastoma and that the expression is increased from several proviruses as compared to normal liver controls. Our results suggest that HERV-K mRNA expression may find use as a biomarker in hepatoblastoma, given the large differential expression profiles in hepatoblastoma, with very low mRNA levels in liver control samples.

Intron retention and its impact on gene expression and protein diversity: A review and a practical guide

Wiley Interdisciplinary Reviews - Rna, Oct 18, 2020

Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An incr... more Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output.This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico

Journal of Clinical Virology, Jun 1, 2018

Background-Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing v... more Background-Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses). In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. The use of Maraviroc is reserved for salvage therapy in South Africa. Objective-In this study, we examined the frequency of R5 and X4 viruses, using next generation sequencing, in patients under treatment to draw inferences on the utility of Maraviroc in a South African population. Study design-Proviral DNA was isolated from peripheral blood mononuclear cells (PBMC) of 72 chronically HIV infected patients on antiretroviral treatment. HIV V3 loop gene was amplified and sequenced on an Illumina MiniSeq platform. Viral subtypes were determined by the jumping profile Hidden Markov Model (jpHMM) and REGA genotyping tools. De Novo consensus sequences were derived for the majority and minority populations for each patient using Geneious ® software version 8.1.5. HIV-1 tropism was inferred using PSSMsinsi, Geno2pheno and Phenoseq-C web-based tools. Results-Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients exclusively *

BMC Medical Genetics, Jan 19, 2019

The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, ... more The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. Methods: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3. 0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. Results: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. Conclusions: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .

Molecular Biology of the Cell, Dec 1, 2016

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is ess... more The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes. Monitoring Editor A

Current HIV Research, 2020

Background: To complete its replication cycle, HIV-1 requires the nucleocytoplasmic export of int... more Background: To complete its replication cycle, HIV-1 requires the nucleocytoplasmic export of intron-containing viral mRNAs. This process is ordinarily restricted by the cell, but HIV overcomes the block by means of a viral protein, Rev, and an RNA secondary structure found in all unspliced and incompletely spliced viral mRNAs called the Rev Response Element (RRE). In vivo activity of the Rev-RRE axis requires Rev binding to the RRE, oligomerization of Rev to form a competent ribonucleoprotein complex, and recruitment of cellular factors including Crm1 and RanGTP in order to export the targeted transcript. Sequence variability is observed among primary isolates in both Rev and the RRE, and the activity of both can be modulated through relatively small sequence changes. Primary isolates show differences in Rev-RRE activity and a few studies have found a correlation between lower Rev-RRE activity and slower progression of clinical disease. Lower Rev-RRE activity has also been associat...

Background: The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. The... more Background: The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. Results: In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcR...

AIDS research and human retroviruses, Jan 4, 2016

The HIV-1 replication cycle requires the nucleocytoplasmic export of intron-containing viral RNAs... more The HIV-1 replication cycle requires the nucleocytoplasmic export of intron-containing viral RNAs, a process which is ordinarily restricted. HIV overcomes this by means of the viral Rev protein, which binds to an RNA secondary structure called the Rev Response Element (RRE) present in all unspliced or incompletely spliced viral RNA transcripts. The resulting mRNP complex is exported through interaction with cellular factors. The Rev/RRE binding interaction is increasingly understood to display remarkable structural plasticity, but little is known about how Rev/RRE sequence differences affect functional activity. To study this issue, we utilized a lentiviral vector assay in which vector titer is dependent on the activity of selected Rev/RRE pairs. We found that Rev/RRE functional activity varies significantly (up to 24-fold) between naturally occurring viral isolates. The activity differences of the Rev/RRE cognate pairs track closely with Rev, but not with RRE activity. This variati...

Identification of a gene encoding for the human formyl peptide receptor

Biochemistry international, 1992

The neutrophil FMLP receptor is involved in activation and subsequent response to certain chemota... more The neutrophil FMLP receptor is involved in activation and subsequent response to certain chemotactic stimuli. The normal receptor has been reported to consist of several components, ranging in size from 43-94 kDa, and to contain both high and low affinity states. However, limited information is available on the gene/s which encode for the receptor. In this study, we have generated oligonucleotide probes derived from a published cDNA sequence encoding for one of the components of the FMLP receptor, and used these probes to amplify genomic DNA from HL-60 cells as well as normal human neutrophils, using the polymerase chain reaction. Such procedure resulted in the amplification of a single, approximately 1 kb fragment of genomic DNA identical in sequence to the cDNA described in the literature for one of the isoforms of the receptor. This finding supports the notion that the human FMLP receptor is encoded by at least one, intronless gene.

A 5’ Splice Site is Essential for REV and REX Regulation of HIV Envelope Protein mRNA Expression

Advances in Molecular Biology and Targeted Treatment for AIDS, 1991

The organization of the HIV genome is much more complex than that of most other retroviruses (for... more The organization of the HIV genome is much more complex than that of most other retroviruses (for a review see (1)). This is due to the presence of several regulatory genes in addition to the structural genes gag, pol and env. This complexity is reflected by the large number of different subgenomic mRNAs found in HIV infected cells (2). A proper balance between these mRNAs has to be achieved to ensure appropriate expression of the different viral proteins.

Interaction of HIV-1 and human salivary mucins

Journal of acquired immune deficiency syndromes, 1994

Previous studies have suggested that salivary secretions may act as inhibitors of HIV-1 replicati... more Previous studies have suggested that salivary secretions may act as inhibitors of HIV-1 replication in vitro. This inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands, and subsequent electron micrographs revealed the association of viral particles with the salivary sediment. Fractionation of human submandibular-sublingual (HSMSL) saliva by size-exclusion chromatography was initiated, and resulting fractions were tested for their ability to modulate the replication of HIV-1 using a plaque assay on HeLa CD4+ cell monolayers. Results indicated that the filtration-sensitive inhibitory activity was primarily associated with the mucin-rich fractions, and the inhibitory activity was found to reduce the number of infectious units by 75%. To determine the identity of the salivary components involved, adsorption experiments involving the interaction of HIV particles with immobilized salivary components were perform...

Experimental Parasitology, 1993

AIDS Research and Human Retroviruses, 1995

We have developed a simple and rapid procedure for the purification of large amounts of Rev prote... more We have developed a simple and rapid procedure for the purification of large amounts of Rev protein over- expressed in E. coli. The purification method, which does not require denaturation of the protein, takes ad- vantage of the positively charged nature of Rev and the ability of Rev to interact with nucleic acids. The pu- rified protein was used to develop three novel murine monoclonal antibodies against Rev. Using fusion proteins between glutathione S-transferase (GST) and various fragments of the Rev protein, we mapped the specificity of these antibodies to different regions of the Rev protein. One antibody, 3H6, is directed against the maleo- lar localization/RRE-binding domain of Rev between amino acids 38 and 44. Another antibody, 3G4, recog- nizes an epitope between amino acids 90 and 116 of Rev. A third antibody, 2G2, does not recognize any of the fusion proteins, and may be directed against a conformational epitope. All three antibodies are able to de- tect Rev on Western blots and to immunoprecipitate Rev under native conditions. However, only 3H6 and 3G4 immunoprecipitate Rev under denaturing conditions and are able to detect Rev expressed in transfected cells by indirect immunofluorescence. These antibodies should prove useful in further studies of Rev function.

Regulation of HIV expression

AIDS, 1991

The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are presen... more The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci (4p16.1b, 22q11.23 and most significantly 3q12.3) accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3' RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec. The HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes.

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.

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.

Journal of Biological Chemistry, Jul 1, 2004