Hervé Perron

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

The multiple sclerosis-associated retrovirus (MSRV) isolated from plasma of MS patients was found to be phylogenetically and experimentally related to human endogenous retroviruses (HERVs). To characterize the MSRV-related HERV family and to test the hypothesis of a replication-competent HERV, we have investigated the expression of MSRV-related sequences in healthy tissues. The expression of MSRV-related transcripts restricted to the placenta led to the isolation of overlapping cDNA clones from a cDNA library. These cDNAs spanned a 7.6-kb region containing gag, pol, and env genes; RU5 and U3R flanking sequences; a polypurine tract; and a primer binding site (PBS). As this PBS showed similarity to avian retrovirus PBSs used by tRNATrp, this new HERV family was named HERV-W. Several genomic elements were identified, one of them containing a complete HERV-W unit, spanning all cDNA clones. Elements of this multicopy family were not replication competent, as gag and pol open reading frames (ORFs) were interrupted by frameshifts and stop codons. A complete ORF putatively coding for an envelope protein was found both on the HERV-W DNA prototype and within an RU5-env-U3R polyadenylated cDNA clone. Placental expression of 8-, 3.1-, and 1.3-kb transcripts was observed, and a putative splicing strategy was described. The apparently tissue-restricted HERV-W long terminal repeat expression is discussed with respect to physiological and pathological contexts.

Cationic amphiphiles have been shown to mediate gene transfer to eukaryotic cells, although the nature and fate of the lipid-DNA complexes is still a matter of debate. Negative staining transmission electron microscopy (TEM) of the complexes in physiological medium, as well as thin-section TEM of transfected cells has been used to visualize the particles and the possible pathways leading to transgene expression. Lipopolyamines form a network of tubular micelles into which plasmid DNA is intertwined and condensed; the cationic particles contain hundreds of plasmid molecules and are heterogeneous with respect to size (0.1-0.5 microgram) and shape. Adherent cells (293M, 3T3, MRC5, primary leptomeningeal cells) take them up readily within minutes by spontaneous endocytosis. Among suspension cells, lymphocytes only incidentally show cytoplasmic inclusions and monocytes degrade the particles by phagocytosis. The marked decrease in transfection efficiency generally observed between adherent and nonadherent cells is thus due to reduce cell binding. This suggests that cationic particles bind to membrane components responsible for Ca2+-mediated cell anchoring to the extracellular matrix. Cation/anion-mediated endocytosis leads to endosomes that are entirely filled with the particles. Consequently, two escape mechanisms may operate: disruption of the lamellar envelope in close contact with tubular micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture. Even for moderately transfected MRC5 cells, 10(2)-10(3) particles are found either free or in cytoplasmic vacuoles 24 h after transfection, highlighting a very inefficient nuclear translocation process. Such high numbers are also the clue to the small concentration window between transfection and cytotoxicity that is often observed with nonviral vectors. Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exchange in the cytoplasm or in the nucleus. The still lower efficiency of free plasmid translocation to the nucleus suggests an active role for the cationic lipid during this step. Although the last stages of the transfection mechanism remain unclear, the present work shows that the major barrier which hampers in vitro gene delivery with cationic vectors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors.

The causes of multiple sclerosis and amyotrophic lateral sclerosis have long remained elusive. A new category of pathogenic components, normally dormant within human genomes, has been identified: human endogenous retroviruses (HERVs). These represent ∼8% of the human genome, and environmental factors have reproducibly been shown to trigger their expression. The resulting production of envelope (Env) proteins from HERV-W and HERV-K appears to engage pathophysiological pathways leading to the pathognomonic features of MS and ALS, respectively. Pathogenic HERV elements may thus provide a missing link in understanding these complex diseases. Moreover, their neutralization may represent a promising strategy to establish novel and more powerful therapeutic approaches.

Multiple sclerosis-associated retroviral element (MSRV) is a retroviral element, the sequence of which served to define the W family of human endogenous retroviruses. MSRV viral particles display proinflammatory activities both in vitro in human mononuclear cell cultures and in vivo in a humanized SCID mice model. To understand the molecular basis of such properties, we have investigated the inflammatory potential of the surface unit of the MSRV envelope protein (ENV-SU), the fraction that is poised to naturally interact with host cells. We report in this study that MSRV ENV-SU induces, in a specific manner, human monocytes to produce major proinflammatory cytokines through engagement of CD14 and TLR4, which are pattern recognition receptors of primary importance in innate immunity. ENV-SU could also trigger a maturation process in human dendritic cells. Finally, ENV-SU endowed dendritic cells with the capacity to support a Th1-like type of Th cell differentiation. The data are discussed in the context of immune responses and chronic proinflammatory disorders.