Science--a life fully lived: Joe Sodroski wins the 2006 Retrovirology Prize.
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Awards and Prizes↗
Biomedical subjects
Andrew M L Lever
Publications and source records attributed to Andrew M L Lever.
An update on malaria prevention, diagnosis and treatment for the returning traveller.
The diagnosis of malaria needs to be considered for every returning traveller with a fever. Compliance with prevention, both pharmaceutical and non-pharmaceutical, is essential for every traveller. New tests for diagnosis are now available. Treatment options have recently expanded to include the artemisinin derivatives that used to be unavailable in the western countries.
Antimalarials↗
Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging.
An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev-loop A interaction supplementary to or preceding that of the Rev-RRE interaction. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev-loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30%. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98% conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.
Animals↗
Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation.
The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.
5' Untranslated Regions↗
Inhibition of HIV-1 replication by oligonucleotide analogues directed to the packaging signal and trans-activating response region.
HIV possesses a remarkable capacity for mutational escape from therapeutics that target the viral proteins and enzymes. Inhibitory strategies aimed at highly conserved nucleic acid sequences within the genome are an attractive alternative. However, it has proven difficult to achieve an effective level of therapeutic at the appropriate site within the cell. Oligonucleotide delivery is a rapidly advancing field. We have investigated oligonucleotide analogues as steric-block therapeutics against two highly conserved regions of the HIV-1 genome. In the study we show that 2'0-methyl/locked nucleic acid oligonucleotides against the packaging signal and trans-activating response regions of HIV can inhibit replication of the virus.
Animals↗
Anti-HIV activity of steric block oligonucleotides.
The unabated increase in spread of HIV infection worldwide has redoubled efforts to discover novel antiviral and virucidal agents that might be starting points for clinical development. Oligonucleotides and their analogs targeted to form complementary duplexes with highly conserved regions of the HIV RNA have shown significant antiviral activity, but to date clinical studies have been dominated by RNase H-inducing oligonucleotide analog phosphorothioates (GEM 91 and 92) that have specificity and efficacy limitations. However, they have proven the principle that oligonucleotides can be safe anti-HIV drugs. Newer oligonucleotide analogs are now available, which act as strong steric block agents of HIV RNA function. We describe our ongoing studies targeting the HIV-1 trans-activation responsive region (TAR) and the viral packaging signal (psi) with steric block oligonucleotides of varying chemistry and demonstrate their great potential for steric blocking of viral protein interactions in vitro and in cells and describe the first antiviral studies. Peptide nucleic acids (PNA) disulfide linked to cell-penetrating peptides (CPP) have been found to have particular promise for the lipid-free direct delivery into cultured cells and are excellent candidates for their development as antiviral and virucidal agents.
Anti-HIV Agents↗
Replication of human immunodeficiency virus type 1 from entry to exit.
It has been 25 years since the recognition of the disease acquired immunodeficiency syndrome (AIDS) in homosexual men in the United States. Much molecular progress in understanding the replication of the human immunodeficiency virus (HIV) has been advanced. Here, we review in a nonexhaustive manner our current knowledge of the replication of HIV-1 inside human cells from entry to exit of the virus.
HIV Infections↗
Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage.
BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.
Animals↗
Blockade of chemokine-induced signalling inhibits CCR5-dependent HIV infection in vitro without blocking gp120/CCR5 interaction.
BACKGROUND: Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection. RESULTS: In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 +/- 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells. CONCLUSION: These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.
Cell Line↗
Identification and visualization of the dimerization initiation site of the prototype lentivirus, maedi visna virus: a potential GACG tetraloop displays structural homology with the alpha- and gamma-retroviruses.
Dimerization of retroviral genomic RNA is essential for efficient viral replication and is mediated by structural interactions between identical RNA motifs in the viral leader region. We have visualized, by electron microscopy, RNA dimers formed from the leader region of the prototype lentivirus, maedi visna virus. Characterization by in vitro assays of the domains responsible for this interaction has identified a 20 nucleotide sequence that functions as the core dimerization initiation site. This region is predicted to form a GACG tetraloop and therefore differs significantly from the kissing loop palindromes utilized to initiate dimerization in primate lentiviruses. The motif is strongly conserved across the ovine and caprine lentiviruses, implying a critical functional role. Furthermore, the proposed GACG tetraloop exhibits marked structural homology with similar structural motifs present in the leader regions of the alpha- and gamma-retroviruses, and the maedi visna virus dimer linkage region is capable of forming heterodimeric species with the Moloney murine leukemia virus Psi domain. This may be indicative of commonality of origin of the two viruses or convergent evolution.
Animals↗
A husband and wife with left hand rashes.
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Exanthema↗
Antiviral activity of steric-block oligonucleotides targeting the HIV-1 trans-activation response and packaging signal stem-loop RNAs.
Mixmer oligonucleotides consisting of residues of both 2'-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5'-UTR of HIV-1 RNA, the transactivation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR beta-Gal cells and subsequently infected with HIV-1.
5' Untranslated Regions↗
HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET.
The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.
Animals↗
The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs.
The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.
5' Untranslated Regions↗
Hepatocyte-specific gene expression from integrated lentiviral vectors.
BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.
Artificial Gene Fusion↗
Human immunodeficiency virus type 2 Gag interacts specifically with PRP4, a serine-threonine kinase, and inhibits phosphorylation of splicing factor SF2.
Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase. Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays. The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag. PRP4 is not incorporated into virus particles. HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2. This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1. Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses. We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2. At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.
Animals↗
Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA.
The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.
5' Untranslated Regions↗
Lentiviral vectors for gene delivery to normal and demyelinated white matter.
Lentiviral vectors are increasingly used for gene delivery to neurons and in experimental models of neurodegeneration. Their use in gene delivery to white matter and their potential value in preventing or repairing CNS demyelination has received less attention. Here we show using a VSV-G-pseudotyped HIV-derived vector expressing the marker gene LacZ that lentiviral vectors transduce the major macroglial cell types present in normal white matter (astrocytes, oligodendrocytes, and oligodendrocyte progenitors). Injection of lentiviral vectors causes an inflammatory response at the injection site characterized by OX42(+) and ED1(+) macrophages, but only a few CD8(+) and no CD4(+) lymphocytes, and mild demyelination. Injection of lentiviral vectors into areas of toxin-induced demyelination resulted in significant numbers of cells expressing the marker gene and was a more effective means of gene delivery than was a LacZ-expressing murine retroviral vector.
Animals↗