146

Literature MOLECULAR MEDICINE TODAY, APRIL 1999 (VOL. 5)

1357-4310/99/$ - see front matter © 1999 Elsevier Science. All rights reserved.

Alphaviruses to the rescue?The ability to genetically engineer animal viruses en-ables their application to medical research. The devel-opment of non-retroviral RNA viruses as vectors to de-liver foreign genes for vaccination and gene therapy isparticularly interesting, especially the domestication ofthe alphavirus group. Two papers in this field have recently been published1,2.

Alphaviruses offer several advantages as expres-sion vectors: they have a broad host range, high levelsof expression and a small genome that is easy to ma-nipulate. Vectors based on Semliki Forest virus (SFV),Sindbis virus and Venezuelan equine encephalitishave been constructed and deliver and express theircargo efficiently. However, the generation of wild-typevirus, owing to recombination between the artificialgenome and a helper genome, remains a problem inmany recombinant viral systems. This biosafety issueis a concern, especially if vectors are to be used invaccine delivery, and methods have been developedto reduce this risk. In the SFV system, Smerdou andLiljeström1 have prevented the production of wild-typevirus by using two, independent helper RNAs; this sys-tem also circumvents the requirement of a viral pro-tease and this portion of the genome has been mu-tated, conferring additional biosafety. Thus, at leasttwo recombination events and one reversion are re-quired to generate replication-competent virus par-ticles. Extensive analysis has failed to demonstrate thepresence of wild-type viruses, emphasizing thebiosafety of the two-helper system.

The expression strategy of alphaviruses imposes asevere burden on normal cellular biochemistry and in-fected cells die quickly, restricting the use of these vec-tors when long-term gene expression is required.However, Agapov et al.2 have developed noncytotoxicSindbis virus vectors, which produce large amounts offoreign protein, by transfecting Sindbis virus repliconsthat express a puromycin N-acetyltransferase intocells grown in the presence of puromycin. Cells thatsurvive in the presence of the drug carry replicons thatare noncytotoxic and express the puromycin-inacti-vating enzyme. Such replicons can then be engineeredto express other foreign genes for long periods in anoncytotoxic manner, which could be useful in bothvaccinology and in the development of gene therapy.

1 Smerdou, C. and Liljeström, P. (1999) Two-helperRNA system for production of recombinant SemlikiForest virus particles, J. Virol. 73, 1092–1098

2 Agapov, E.V. et al. (1998) Noncytopathic Sindbisvirus RNA vectors for heterologous gene expres-sion, Proc. Natl. Acad. Sci. U. S. A. 95, 12689–12994

Roger Hewsonroger.hewson@camr.org.uk

Drug or poison:dosage is everythingFabry disease is a lysosomal storage disorder,which is caused by a deficiency of a lysosomalenzyme, a-galactosidase A (a-Gal A) that results in the abnormal metabolism of glyco-sphingolipids. Fabry patients suffer from renalfailure and premature cardiac arrest; no ef-ficient treatment of this disease is currentlyavailable. In Fabry patients carrying the R301Qmutation in a-Gal A, the mutant enzyme retainsan intact catalytic center but this is improperlyfolded. As a result, it gets ‘trapped’ in the endoplasmic reticulum and does not reach itsfinal destination, the lysosome. When studyingthe effects of a potent inhibitor of a-Gal A,1-deoxy-galactonojirimycin (DGJ), Fan et al.1

made a surprising discovery: when adminis-tered at lower doses, DGJ did not inhibit but en-hanced a-Gal A activity in lymphoblasts fromR301Q Fabry patients. Using reverse tran-scription (RT)-PCR and western-blot analyses,they found that in lymphoblasts cultured withDGJ, the amount of a-Gal A mRNA was unchanged but the amount of mature protein

was substantially increased and more of itwas transported to the lysosomes. This sug-gests that at lower doses DGJ acts as a mol-ecular chaperone and forces the mutant en-zyme to adopt the proper configuration. DGJwas then administered to transgenic mice ex-pressing the R301Q mutant enzyme. Theconsumption of the drug resulted in a several-fold increase of a-Gal A activity in the heartand kidney without any toxic effects to thetreated animals. These results suggest thatDGJ could be a long-awaited treatment ofFabry disease; because several DGJ deriva-tives are known potent inhibitors of other gly-cosidases, this type of drug could become anew paradigm to treat other metabolic dis-orders. These findings support an old adage:dosage is everything.

1 Fan, J-Q. et al. (1999) Accelerated transportand maturation of lysosomal a-galactosi-dase A in Fabry lymphoblasts by an enzymeinhibitor, Nat. Med. 5, 112–115

Eugene Ivanov PhD eivanov@aol.com

CAG expansions encoding polyglutaminetracts have been identified as the causativemutations in eight neurodegenerative dis-eases, including Huntington’s disease (HD).These disorders are characterized by intra-cellular protein inclusions that contain thepolyglutamine expansion. Cellular toxicity isdetermined by the number of glutamines but itis also influenced by fragment size. Inclusionsare predominantly nuclear, however, cyto-plasmic deposits have also been detected inHD brains. Hackam et al.1 have investigatedthe link between the subcellular localization ofaggregates and toxicity in HD using con-structs that express fragments of mutanthuntingtin in vitro. Protein was localized usingimmunofluorescence microscopy and toxicitywas assayed by measuring susceptibility toapoptotic stress. They found that the putativenuclear localization signal (NLS) of huntingtinis non-functional so they added a functionalNLS to large fragments and a nuclear exportsequence to shorter fragments to reverse

their normal localizations. Irrespective of frag-ment size, and regardless of the length of thepolyglutamine tract, they found no significantdifferences in the toxicity of nuclear or cyto-plasmic aggregates. This work opens up thedebate on the role of intraneuronal inclusionsin disease pathogenesis as others have re-ported that cytotoxicity is exclusively nuclear2.These issues are of paramount importance ifwe are to target therapeutics effectively.

1 Hackam, A.S. et al. (1999) In vitro evidencefor both the nucleus and cytoplasm as subcellular sites of pathogenesis inHuntington’s disease, Hum. Mol. Genet. 8,25–33

2 Saudou, F. et al. (1998) Huntingtin acts in thenucleus to induce apoptosis but death doesnot correlate with the formation of intra-nuclear inclusions, Cell 95, 55–66

Sarah Lloyd PhDs.lloyd@ic.ac.uk

Huntingtin toxicity: nucleus or cytoplasm?

Back

grou

nd im

age

repr

oduc

ed fr

om R

ef. 1

, with

per

mis

sion

from

Oxf

ord

Uni

vers

ity P

ress

.