Ultrastructural and Biochemical Analysis of In Vivo and In Vitro Capsid Assembly of the Alkaliphilic Phage [Phi] 1N2-2 for Applications in Nanomedicine

Citation:
Rothschild L, Mwaura F, Kabaru J, Lobo N, Moulton K, Lobo C, Duboise S. "Ultrastructural and Biochemical Analysis of In Vivo and In Vitro Capsid Assembly of the Alkaliphilic Phage [Phi] 1N2-2 for Applications in Nanomedicine." Microscopy and Microanalysis. 2012;18(2):110.

Abstract:

Bacteriophage 1N2-2 infects a narrow range of alkaliphilic bacterial strains phylogenetically
related to the family Idiomarinacea [1]. Both phage and host bacterium were isolated from alkaline
and saline waters of Lake Nakuru, a soda lake of Kenya’s Great Rift Valley. Virion structures
including capsids must tolerate extreme alkaline environments of pH 10 and above. The phage is
morphologically and genetically related to lambdoid viruses of the family Siphoviridae (Fig. 1B)[1].
Genomic analysis revealed that 1N2-2 genes are organized in functional modules. Capsid
morphogenesis genes are homologous both in order and predicted amino acid sequence to the
corresponding genes of the coliphage HK97 (Fig. 1A )[2]. Like many dsDNA viruses, HK97
assembles multiple subunits of a single gene product into a protocapsid around a transient
protein-scaffolding core that is subsequently cleaved by a protease at the time of DNA packaging
triggering expansion and stabilization of the mature capsid [3]. 1N2-2, like HK97, lacks an
independent capsid scaffolding gene but rather, as we have inferred, the N-terminal 110 residues of
the major head protein (MHP) encoded by gene 10 probably assume a coiled-coil folding typical of
scaffolding proteins.
We cloned the 1N2-2 gene 10 MHP sequence into a pQE-Tri-system plasmid (Qiagen) and have
expressed a 46-kDa MHP protein in Escherichia coli strain SG19003 (Qiagen) that can be
precipitated by 8% PEG treatment in ice and low speed centrifugation (Fig. 1C and 1E).
Transmission electron microscopy (TEM) analysis shows the presence of virus-like particles (VLP)
of similar size and shape as empty proheads of naturally formed 1N2-2 (Fig. 1B). Alternatively,
the 3’ end of the gene 10 in the pQE vector was modified to eliminate the stop codon allowing in
frame translation of ten additional codons including eight histidine residues at the C-terminus of
MHP (MHP-his). Based on a Swiss-model prediction of the 1N2-2 MHP protein sequence using
the structure of phage HK97 MHP as template (PDB 1OHG)[4] we inferred that the C-terminus and
its additional modifications would be located on the surface of the recombinant capsid allowing the
purification of fully assembled capsids with Ni-NTA affinity chromatography (data not shown).
SDS-PAGE analysis and UAc-stained protein samples visualized by TEM show that MHP-his
expressed in E. coli is a protein of 46.5 kDa (Fig. 1E) that can assemble into VLPs, possibly
proheads of 46 nm in diameter (Fig. 1D). Other non-closed structures that are seen in the sample are
presumably incomplete capsids (Fig 1 C and 1D). We are currently investigating whether capsid
morphogenesis including incomplete structures and proheads produced from the expression plasmid
in E. coli differ from natural capsid morphogenesis in the alkaliphilic host of 1N2-2. We are
formulating capsid assembly buffers to produce fully closed stable VLP in vitro. We conclude that the Φ1N2-2 MHP protein, either intact or with histidineresidues at the C-terminus, is sufficient to produce VLP in theabsence of other virally-encoded proteins. Weexpectthe MHP C-terminus can be used to
display antigens on the prohead surface while modifications of the N-terminus may be used for
packaging antigens internally providing a versatile VLP platform for vaccine development and
other nanomedicine applications.

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