Sondra Schlesinger

Alphavirus vectors for gene expression and vaccines.

Alphavirus expression vectors are finding novel uses in research. They are showing increasing promise as vaccines and are being developed for diagnostic assays of other viruses. Some highlights over the past couple of years include improvements in packaging of replicons, targeting of Sindbis virus replicons, stable cell lines that can be induced to produce replicons, and the isolation of noncytopathic variants of Sindbis virus replicons. Reports that alphavirus vectors can efficiently infect neurons in rat hippocampal slices should increase their use in neurobiological studies.

Alphaviruses — vectors for the expression of heterologous genes

Abstract DNA viruses and retroviruses are well established as vectors for the expression of heterologous genes, but there is increasing interest in the possibilities of using RNA viruses, which do not replicate through a DNA intermediate, for this purpose. This article summarizes some of the general properties of RNA viruses and concentrates on one class of RNA viruses — the alphaviruses — and their potential as expression vectors.

Antigenic characterization of two sindbis envelope glycoproteins separated by isoelectric focusing.

Abstract The isoelectric point of intact Sindbis virus was determined to be pI 4.2 by using isoelectric focusing in sucrose gradients. Following nonionic detergent disruption and subsequent electrofocusing, three structural proteins were separated. The two envelope glycoproteins (E1 and E2) were isolated at isoelectric points pI 6 and 9, respectively, and nucleocapsid protein was localized toward the anode at approximately pH 3. The pI 6 protein appeared to be the only glycopeptide with hemagglutinating activity, while both pI 6 and 9 proteins were antigenic in complement fixation and radioimmune precipitation tests. The pI 6 (E1) protein cross-reacted with antisera to the closely related western equine encephalitis virus. In contrast, the pI 9 (E2) protein antigen appeared Sindbis virus specific. Only antiserum prepared to the pI 9 protein antigen neutralized infectious Sindbis virus.

Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein.

The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.

A role for oligosaccharides in glycoprotein biosynthesis

Abstract Oligosaccharide chains can influence the ability of a protein to fold properly. The large size of the precursor of asparagine-linked oligosaccharides may be essential if certain proteins are to achieve the correct tertiary structure.

Alphavirus vectors: development and potential therapeutic applications.

Alphaviruses are RNA enveloped viruses that are proving their value as expression vectors. They are particularly well-suited for this role as they are easily and quickly engineered and can be used to produce high levels of proteins of interest. A promising and important use is as vaccines against disease-causing agents such as HIV. The three alphaviruses now serving as vectors are Sindbis virus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEE) virus. Sindbis virus and SFV are well-known models for studies in molecular and cell biology; VEE virus is a human pathogen and had received some previous notoriety as a potential biological weapon. It is now becoming a potentially valuable vaccine vector. All three viruses are being tested as vaccines but, at present, only Sindbis virus and SFV have been considered for other uses. Sindbis virus vectors have been developed to screen libraries for the identification of new proteins and to devise sensitive assays to detect viruses more difficult to grow in culture. Both Sindbis virus and SFV vectors are serving as tools for fundamental studies in biology, examples include development in insects and analysis of protein functions in neuronal cells. In this article the replication strategy of alphaviruses and the different ways they can be engineered to serve as expression vectors is described. This provides an introduction to the ways these vectors have been used and illustrates the promise these vectors offer.

Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons.

Sindbis virus-based vectors have been successfully used for transient heterologous protein expression in neurons. Their main limitation arises from infection-associated cytotoxicity, attributed largely to a progressive shut down of host cell protein synthesis. Here we evaluated a modified Sindbis vector, based on a viral strain containing a point mutation in the second nonstructural protein, nsP2 P726S, described to delay inhibition of protein synthesis in BHK cells [Virology 228 (1997) 74], for heterologous expression in neurons in vitro and in vivo. First, we constructed an optimized helper vector, termed DH-BB(tRNA/TE12), for production of SINrep(nsP2S(726)) viral particles with low levels of helper RNA co-packaging and high neurospecificity of infection. Second, we determined that hippocampal primary neurons infected with SINrep(nsP2S(726)) virus expressing EGFP showed a delayed onset of viral induced cytotoxicity and higher levels of EGFP expression in comparison to cells infected with wild type SINrep5 EGFP-expressing virus. However, a strong decrease in protein synthesis still occurred by day 3 postinfection. The SINrep(nsP2S(726)) vector is thus well suited for rapid high level expression within this time window. As an experimental example, we demonstrate the applicability of this system for high-resolution two-photon imaging of dendritic spines in vivo.

Formation and Assembly of Alphavirus Glycoproteins

Among the variety of enveloped viruses that exist in nature, the Togaviridae family is generally considered to be among the simplest with regard to structure and composition of the virus. The virion structure is examined in detail in Chapter 22. In this chapter, we focus on the major protein components of the virion envelope and describe modifications of these proteins that occur as they mature from nascent polypeptides to fully mature and functional glycoproteins that form the spikes of the infectious particle. Most of our information about togavirus glycoproteins comes from biochemical studies of the two alphaviruses, Semliki Forest virus and Sindbis virus (Garoff et al., 1982). These viruses have provided valuable models for the analysis of membrane proteins.

The effects of inhibitors of glucosidase I on the formation of Sindbis virus

We have examined the effects of deoxynojirimycin and castanospermine, compounds known to inhibit the removal of glucose from high mannose asparagine-linked oligosaccharides, on the formation of Sindbis virus. These drugs inhibited virion formation in baby hamster kidney (BHK) cells, 15B - the CHO cell line that lacks GlcNAc transferase activity, and chicken embryo fibroblasts, although our results with the latter cells were variable. We analyzed the [3H]mannose-labeled oligosaccharides from Sindbis virus infected 15B cells. Those from control cells were predominantly GlcNAc2Man5. Oligosaccharides from the treated cells were larger than the Man5 species and as expected, were partially resistant to alpha-mannosidase. The growth of Sindbis virus was inhibited to a much greater extent at 37 degrees C than at 30 degrees C in BHK cells treated with either deoxynojirimycin or castanospermine. Both of these compounds also inhibited the proteolytic cleavage of the viral glycoprotein precursor, PE2, to the virion glycoprotein, E2, but did not prevent the migration of the glycoprotein to the cell surface. These results, taken together with our earlier studies with vesicular stomatitis virus (Schlesinger et al., 1984) provide strong evidence that the removal of glucose residues during the processing of asparagine-linked oligosaccharides is critical for some proteins to achieve a functional conformation.

Submaxillary gland of mouse: properties of a purified protein affecting muscle tissue in vitro.

A protein from the salivary gland of mice has been highly purified. It affects embryonic muscle tissue in vitro and has both esterase and peptidase activities. Addition of the pure protein to tissue culture in synthetic medium causes dissociation of muscle fibers in individual myoblasts with loss of myosin. This biological activity, as well as the esterase activity, is inhibited by low concentrations of phenylmethanesulfonyl fluoride; this suggests that the effect on the tissue is a consequence of the proteins enzymatic activities.