Visualization of feline calicivirus replication in real-time with recombinant viruses engineered to express fluorescent reporter proteins

Abstract

Caliciviruses are non-enveloped, icosahedral viruses with a single-stranded, positive sense RNA genome. Transposon-mediated insertional mutagenesis was used to insert a transprimer sequence into random sites of an infectious full-length cDNA clone of the feline calicivirus (FCV) genome. A site in the LC gene (encoding the capsid leader protein) of the FCV genome was identified that could tolerate foreign insertions, and two viable recombinant FCV variants expressing LC fused either to AcGFP, or DsRedFP were recovered. The effects of the insertions on LC processing, RNA replication, and stability of the viral genome were analyzed, and the progression of a calicivirus single infection and co-infection were captured by real-time imaging fluorescent microscopy. The ability to engineer viable recombinant caliciviruses expressing foreign markers enables new approaches to investigate virus and host cell interactions, as well as studies of viral recombination, one of the driving forces of calicivirus evolution.

The Feline Calicivirus Leader of the Capsid Protein Is Associated with Cytopathic Effect

“…These findings, along with our previous discovery that the LC can tolerate the insertion of foreign sequences twice its own length between amino acids 88 and 89 (1), lead us to propose that the LC contains at least two domains to mediate its function in viral CPE: an N-terminal domain (within aa 1 to 88) that is associated with cell rounding and death and a C-terminal domain (within aa 89 to 124) that is involved in virus spread. Although the precise role of this unique vesivirus protein and its mechanism of action require further investigation, additional study might give insight into how caliciviruses interact with host cells to produce progeny in one of the fastest replication cycles among viruses.…”

“…Feline calicivirus (FCV) is in the genus Vesivirus of the family and has been an important model for studying calicivirus replication because it grows efficiently in cell culture and has a reverse genetics system (1)(2)(3)(4)(5). The RNA genomes of caliciviruses range in size from ϳ6.7 to 8.5 kb and typically encode 8 or 9 viral proteins from two (Sapovirus, Nebovirus, and Lagovirus) or three (Norovirus and Vesivirus) open reading frames (ORFs).…”

“…Transient expression of the LC was reported to enhance replication of a human norovirus RNA replicon (23), and an increase in the level of mRNA for the low-density lipoprotein receptor (LDLR) was observed (24). We showed previously that the FCV LC can tolerate the insertion of foreign proteins such as green fluorescent protein and DsRed between amino acids 88 and 89, and recombinant viruses expressing fluorescent markers were used to visualize a calicivirus infection in real time (1).…”

“…Standard recombinant DNA methods were employed to generate recombinant FCV full-length (FL) clones, as described previously (1,4). To introduce a unique KpnI cleavage site into the 5= end of the FCV VP1 sequence (downstream of the LC and VP1 border), the FL clone pR6 (4) was modified with a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA), using the primer pair 5=-CTGCCCCAGAGCAAGGtACcGTG GTTGGAGGAG (designated Urb-VP1-KpnI_F) and 5=-CTCCTCCAAC CACgGTaCCTTGCTCTGGGGCAG (designated Urb-VP1-KpnI_R).…”

“…First, the mKate2 coding sequence was PCR amplified using the pmKate2-N plasmid as a template (Evrogen, Moscow, Russia) to include the bordering KpnI and AflII restriction sites and a linker sequence (GGSGGS). Then, the PCR-amplified mKate2 was digested, purified, and ligated into the LC transposon insertion site (TIS) of the pR6-LC FL clone between amino acids (aa) 88 and 89, previously identified to tolerate insertions without abrogating the recovery of virus (1). Individual colonies were screened by PCR to identify plasmids that contained the insert.…”