Mobilization of Full-Length Semliki Forest Virus Replicon by Retrovirus Particles

Abstract

Conciliating biosafety with efficient gene transfer remains a constant concern in the development of retroviral vectors. Semliki Forest virus (SFV) replicons allow important retroviral vector production with interesting features. It is noteworthy that retroviruses have the ability to package ⌿ ؉ and, to some extent, ⌿ ؊ cellular RNAs. Therefore, it was important to study the retroviral transfer of highly abundant SFV genomes expressing retroviral proteins. Here, we show that full-length SFV-vector replicons, with or without ⌿, are efficiently packaged into retrovirus particles. Mechanistically, our data suggest that SFV packaging is the sum of its retroviral nucleocapsid-dependent recruitment together with a passive hijacking of membrane-anchored SFV replicon. A direct consequence of this phenomenon is the formation of particles harboring autonomous replicative abilities and contaminating vector preparations. Importantly, we confirm that retroviral SFV mobilization is not an exclusive feature of murine gamma retroviruses, since it is also observed using lentivectors.It is well established that retroviral vectors derived from simple oncoretroviruses, murine leukemia virus (MLV), or lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are effective and versatile gene delivery systems (31). Recombinant particle production involves the expression of two components, either continuously in stable packaging cell lines or transiently by cotransfection of naïve cells (31). Vector production by stable cell lines can be maximized using bioreactors or various chemical boosts (4,14,23,26,32). Most transient production protocols rely on DNA transfection, with the rest involving delivery of retroviral components using heterologous viral vectors (20,31,38,40,43,44). Among these is the Semliki Forest virus (SFV) vector system (20, 43). It was initially developed for robust protein production in mammalian cells (1). SFV-positive RNA is capable of autoamplification through self-primed replication (42). In addition, the SFV genome contains an internal promoter, which prompts a further amplification via transcription of a subgenomic RNA (42). Within vectors, the subgenomic RNA encodes the transgene (25). Li and Garoff and others have developed an SFV system allowing retroviral vector production (20, 43). Interestingly, Li and collaborators have shown that the Semliki-derived system, which displays an exclusive cytoplasmic replication, allows the transfer of intron-containing retroviral vectors (19).An important consideration when using retroviral vectors as therapeutic tools is biosafety (31). Treatment of severe combined immunodeficiency (SCID) patients has emphasized the risk of insertional mutagenesis with retroviral vector based on MLV (12). As a consequence, targeted integration is now a major goal (2). Replication-competent retrovirus (RCR) in vector preparations is also to be considered. In animal models, RCRs have been shown to trigger cancer (3, 13, 24). Using classical retroviral production systems, the emerg...

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

“…The major difference between the study by Piver and co-workers 34 and our current study is that they adopted an SFV-based vector containing a non-functional internal 26S promoter, which should lead to the exclusive production of full-length SFV genomic RNA 49 and a reporter gene under the transcriptional control of the SFV replicase. In contrast, our system is characterized by the SFV replicase, which regulates transcription of the entire cis-acting element of the lentiviral component, that is the HIV-1 vector unit, and by the presence of a wildtype 26S subgenomic promoter.…”

“…Indeed, the use of SFVderived vectors to produce recombinant retroviral particles has been proposed. [30][31][32][33] However, despite the attractive features, this approach has shown that SFV replicons could be packaged within the recombinant particles, 34,41 rendering it unsuitable in gene therapy application for safety reasons. The purpose of this study was to clarify this issue by adopting an SFV/HIV hybrid system for the expression of the HIV-1 vector elements under the transcriptional control of the SFV replicase and for the production of transducing pseudotyped lentiviral particles.…”

“…By using similar amounts of (Figure 5a). Piver and collaborators 34 have recently shown the mobilization of full-length SFV replicons by retroviral particles. Therefore, we investigated the presence of the SFV/Gag-Pol and the SFV/VSV G replicons, as well as that of the SFV/Helper C and the SFV/Helper S replicons, in the supernatant of co-transduced cells by real-time PCR using specific primers for the Gag, the VSV-G, the SFV/Helper C and the SFV/Helper S coding sequences.…”

“…However, even though the expression of the SFV/HIV CAT vector in transfected cells was not particularly high, efficient transducing ability of SFV/HIV CAT particles was achieved. Piver et al 34 have recently shown that SFV vectors are efficiently incorporated into gamma retroviral and lentiviral particles in a retroviral packaging sequenceindependent manner. Our results are in agreement with these findings, as when we analyzed the pseudotyped HIV-1 particles released from co-transduced target cells, SFV/Gag-Pol, SFV/VSV G , as well as SFV/Helper C and SFV/Helper S replicons could be detected.…”

“…Indeed, the use of SFV-derived vectors to produce recombinant gamma retroviruses has been proposed, and it has been shown that RNA-based SFV expression system can be used for the production of Moloney murine leukemia virus particles at high titers. [30][31][32] In this context, it has been reported that SFV replicons could be packaged within recombinant retroviral particles, including murine gamma retrovirus 33,34 and lentivirus, 34 indicating that the production of retroviral particles under SFV-based transcriptional control must be prohibited in gene therapy approaches. In this study, to gain further insights into mobilization of SFV replicons by means of lentiviral particles, we designed a chimeric SFV/HIV vector system that contains both the HIV-1 cis-and transacting elements under the transcriptional control of the SFV replicase and investigated the ability of the hybrid SFV/HIV system to produce recombinant lentiviral particles capable of transducing target cells.…”