The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System

Abstract

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.

Visualization of Retroviral Replication in Living Cells Reveals Budding into Multivesicular Bodies

“…MLV Gag‐CFPAAAA and Gag‐PolAAAA were prepared by site‐directed mutagenesis. Friend MLV Env‐YFP was generated by YFP insertion into the NheI site of the construct pFr‐MuLV273/274 (49) and transfer of the Env gene into pcDNA3. MCAT‐CFP was a variant of MCAT‐GFP (50).…”

“…To produce Env‐YFP‐labeled MLV virions, YFP was cloned into the NheI site of the Friend 57–273/4 construct from Abraham Pinter (New York University, New York, NY, USA) (49). This construct generates a replication‐competent virus, which was produced in a chicken cells stably expressing MCAT‐1 (DFJ8 cells; Stephen Hughes, National Cancer Institute‐Frederick, Frederick, MD, USA) (57).…”

Viruses as Building Blocks for Materials and Devices

“…A number of viral membrane fusion proteins are known to have permissive insertion sites for the introduction of foreign peptide sequences. Examples of these are the membrane fusion proteins from baculovirus,31 avian leukosis virus,32 vesicular stomatitis virus,33 murine leukemia virus,34 influenza A,35 and others. Although the modified viruses maintain their infectivity, it can be diminished if long polypeptides are inserted resulting in steric hindering of the dynamics of the fusion process.…”

Viren als Bauelemente für Materialien und Strukturen

“…Einige virale Membranfusionsproteine weisen permissive Insertionsstellen für das Einfügen zusätzlicher Peptidsequenzen auf. Beispiele dafür sind Membranfusionsproteine von Baculoviren,31 Vogel‐Leukoseviren,32 vesikulären Stomatitisviren,33 murinen Leukämieviren,34 von Influenza A35 und anderen. Obwohl die an ihrer Oberfläche modifizierten Viren ihre Infektiosität behalten, können insertierte Polypeptide die Dynamik des Fusionsprozesses sterisch behindern und somit die Infektivität der Viren reduzieren.…”