Tatyana Kozlovska

Membrane proteins organize a symmetrical virus.

Alphaviruses are enveloped icosahedral viruses that mature by budding at the plasma membrane. According to a prevailing model maturation is driven by binding of membrane protein spikes to a preformed nucleocapsid (NC). The T = 4 geometry of the membrane is thought to be imposed by the NC through one‐to‐one interactions between spike protomers and capsid proteins (CPs). This model is challenged here by a Semliki Forest virus capsid gene mutant. Its CPs cannot assemble into NCs, or its intermediate structures, due to defective CP–CP interactions. Nevertheless, it can use its horizontal spike–spike interactions on membrane surface and vertical spike–CP interactions to make a particle with correct geometry and protein stoichiometry. Thus, our results highlight the direct role of membrane proteins in organizing the icosahedral conformation of alphaviruses.

RNA Phage Qβ Coat Protein as a Carrier for Foreign Epitopes

The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known ‘display vectors’ on the basis of coat proteins of RNA phage group I, group III p

Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods

Icosahedral virus‐like particles (VLPs) of RNA phage Qβ are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between β‐strands F and G (FG loop) of the monomeric subunits, which determine the five‐fold and quasi‐six‐fold symmetry contacts of the VLPs. In order to reduce the stability of Qβ VLPs, we mutationally converted the amino acid stretch 76‐ANGSCD‐81 within the FG loop into the 76‐VGGVEL‐81 sequence. It led to production in Escherichia coli cells of aberrant rod‐like Qβ VLPs, along with normal icosahedral capsids. The length of the rod‐like particles exceeded 4–30 times the diameter of icosahedral Qβ VLPs.

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies

Summary.  Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV‐genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1–48 of the pre‐S1 domain, fused to S (pre‐S1.1–48/S). The antigen loop in S protein and the selected pre‐S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 107 rSFV particles and 108 rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast‐derived S antigen but equally well with patient‐derived S antigen. Immunization with rSFV encoding pre‐S1.1–48/S resulted in formation of pre‐S1‐ and S‐specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre‐S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma‐derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre‐S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre‐S1.1–48/S vector, indicating the strongest Th1 response. This vector type may induce subtype‐independent and S‐escape‐resistant neutralizing antibodies against HBV.