The alphaviruses: gene expression, replication, and evolution.

Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genes in Vitro and Immunization against Heterologous Pathogens in Vivo

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.

Viral vectors for gene transfer: a review of their use in the treatment of human diseases.

Most plant viruses are absolutely dependent on a vector for plant-to-plant spread. Although a number of different types of organisms are vectors for different plant viruses, phloem-feeding Hemipterans are the most common and transmit the great majority of plant viruses. The complex and specific interactions between Hemipteran vectors and the viruses they transmit have been studied intensely, and two general strategies, the capsid and helper strategies, are recognized. Both strategies are found for plant viruses that are transmitted by aphids in a nonpersistent manner. Evidence suggests that these strategies are found also for viruses transmitted in a semipersistent manner. Recent applications of molecular and cell biology techniques have helped to elucidate the mechanisms underlying the vector transmission of several plant viruses. This review examines the fundamental contributions and recent developments in this area.

Virus-Vector Interactions Mediating Nonpersistent and Semipersistent Transmission of Plant Viruses

In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface.

A DNA comprises a cDNA clone coding for an infectious Venezuelan Equine Encephalitis Virus RNA transcript and a heterologous promoter sequence positioned upstream from the cDNA clone and operatively associated therewith. A method of making a live attenuated Togavirus useful as a vaccine, and cDNA clones which code for attenuated Togaviruses, is also disclosed.

cDNA clone coding for Venezuelan equine encephalitis virus and attenuating mutations thereof

Topographic analysis of tobacco etch virus capsid protein epitopes.

In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant and mutations affecting virulence.

L. V. Willis

Papers

In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface.

cDNA clone coding for Venezuelan equine encephalitis virus and attenuating mutations thereof

Topographic analysis of tobacco etch virus capsid protein epitopes.

In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant and mutations affecting virulence.