Semliki Forest Virus Expression System: Production of Conditionally Infectious Recombinant Particles

Abstract

In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12

“…6,7 Semliki Forest virus (SFV), an alphavirus, is an enveloped positive-sense RNA virus of the family Togaviridae; it has been developed into a transient RNA-based suicidal expression vector system. 8 This system consists of a vector in which foreign genes can be expressed, and two helper vectors that encode the structural protein genes. Cotransfection of all three vectors into cells results in release of recombinant virus-like particles (VLPs) coding for the foreign gene.…”

Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines

“…[15][16][17][18] SFV is a positive-strand RNA virus belonging to the genus Alphaviruses of the family Togaviridae. 19 Alphaviruses replicate in the host cell cytosol without integration of the viral genetic material in the cellular genome.…”

“…The other subgenomic RNA lacked the packaging signal and encoded the structural proteins with a mutation in the p62 gene. 15 The mutation in p62 prevents cleavage of precursor p62 into E2 and E3. Therefore, generated stocks of virus contained non-infectious virions with precursor protein p62 in the envelope.…”

“…Immediately before transduction, particles were activated by cleaving the precursor with chymotrypsin that liberated the fusion active E2 protein. 15 Twenty-four hours after electroporating RNA into BHK cells, particles were harvested. Using the procedures described in Materials and methods, SFV particle-stocks were obtained with titers ranging from 2 × 10 8 to 3 × 10 9 infectious U/ml.…”

Semliki Forest virus-based DNA expression vector: transient protein production followed by cell death

“…Some of these self-amplifying systems use RNA genomes of alphaviruses, such as Sindbis virus [1][2][3] and Semliki Forest virus (SFV). 4,5 SFV, a member of the Alphavirus genus, is a smallenveloped virus with a single-stranded RNA genome of positive polarity. The 5′ two-thirds of the viral genome encode nonstructural (replication) proteins (nsPs1-4) and the 3′ one-third encodes structural proteins.…”