Selective transport of fluorescent proteins into the phage nucleus

Abstract

Upon infection of Pseudomonas cells, jumbo phages 201Φ2-1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.

Viral speciation through subcellular genetic isolation and virogenesis incompatibility

“…We identified five potential nucleases from ΦPA3 and imported them into the ΦKZ nucleus by taking advantage of our fortuitous finding that GFPmut1 and any proteins fused to it, are imported into the nucleus of ΦKZ but not ΦPA3 (Fig. 4c) 20 . In contrast, sfGFP does not alter protein localization and naturally resides in the cytoplasm ( Fig.…”

“…In contrast, sfGFP does not alter protein localization and naturally resides in the cytoplasm ( Fig. 4c), serving as a negative control 20 .…”

Architecture and self-assembly of the jumbo bacteriophage nuclear shell

“…The pHERD-30T plasmid was used for expressing eGFP-tagged full-length and truncated Chimallin in P. chlororaphis 22,23 . All constructs were designed with eGFP fused to the N terminus and synthesized by GenScript.…”

Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage

“…Immediately upon injection of phage DNA into the host cell, several large bacteriophages including related PhiKZ-like Pseudomonas phages PhiPA3, 201Phi2-1, and PhiKZ, as well as Serratia phage PCH45, enclose their replicating DNA in a roughly spherical, nucleus-like, proteinaceous shell that expands as the viral DNA replicates (11)(12)(13)(21)(22)(23). The phage nucleus compartmentalizes proteins according to function, with proteins involved in DNA replication, recombination, and transcription localized inside and those involved in translation and metabolic processes outside (11,12,24). This compartmentalization provides an advantage to nucleus-forming phages by allowing them to shield their DNA from host-encoded DNA restriction and CRISPR-Cas systems (13,25).…”