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Alphavirus Viral Vectors

Alphaviruses, like Sindbis Virus and Semliki Forest Virus, belong to the Togaviridae family of viruses. There are 27 alphaviruses, able to infect various vertebrates such as humans, rodents, birds, and larger mammals such as horses as well as invertebrates. Alphaviruses particles are enveloped have a 70 nm diameter, tend to be spherical and have a 40 nm isometric nucleocapsid.

The genome of alphaviruses consists of a single stranded positive sense RNA. The total genome length ranges between 11 and 12 kb, and has a 5’ cap, and 3’ poly-A tail. There are two open reading frames (ORF’s) in the genome, non-structural and structural. The first is non structural and encodes proteins for transcription and replication of viral RNA, and the second encodes four structural proteins: Capsid protein C, Envelope glycoprotein E1, Envelope glycoprotein E2, and Envelope glycoprotein E3. The expression of these proteins and replication of the viral genome all takes place in the cytoplasm of the host cells.

Figure 1. Surface of an Alphavirus (computer-generated model).

Alphaviruses are of interest to gene therapy researchers, in particular the Ross River virus, Sindbis virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus have all been used to develop viral vectors for gene delivery. Application of replication-deficient vectors leads to short-term expression, which makes these vectors highly attractive for cancer gene therapy. Alphavirus vectors carrying therapeutic or toxic genes used for intratumoral injections have demonstrated efficient tumor regression.

Figure 2. Genome organisation of Semliki Forest virus.

Of particular interest are the chimeric viruses that may be formed with alphaviral envelopes and retroviral capsids. Such chimeras are termed pseudotyped viruses. Alphaviral envelope pseudotypes of retroviruses or lentiviruses are able to integrate the genes that they carry into the expansive range of potential host cells that are recognized and infected by the alphaviral envelope proteins E2 and E1. The stable integration of viral genes is mediated by the retroviral interiors of these vectors.

There are limitations to the use of alphaviruses in the field of gene therapy due to their lack of targeting, however, through the introduction of variable antibody domains in a non-conserved loop in the structure of E2, specific populations of cells have been targeted. Furthermore, the use of whole alphaviruses for gene therapy is of limited efficacy both because several internal alphaviral proteins are involved in the induction of apoptosis upon infection and also because the alphaviral capsid mediates only the transient introduction of mRNA into host cells. Neither of these limitations extend to alphaviral envelope pseudotypes of retroviruses or lentiviruses.

However, the expression of Sindbis virus envelopes may lead to apoptosis, and their introduction into host cells upon infection by Sindbis virus envelope pseudotyped retroviruses may also lead to cell death. The toxicity of Sindbis viral envelopes may be the cause of the very low production titers realized from packaging cells constructed to produce Sindbis pseudotypes.

Another branch of research involving alphaviruses is in vaccination. Alphaviruses are apt to be engineered to create replicon vectors which efficiently induce humoral and T-cell immune responses. They could therefore be used to vaccinate against viral, bacterial, protozoan, and tumor antigens.