Bunyavirus reverse genetics

To date, a number of bunyaviruses have been rescued and these include BUNV

2006), AKV (Ogawa et al., 2007), SBV (Elliott et al., 2013; Varela et al., 2013),

SFTSV (Brennan et al., 2015), UUKV (Rezelj et al., 2015) and CCHFV (Bergeron et

al., 2015). To rescue these viruses mammalian cell lines are transfected with three

“transcription plasmids” that contain an antigenomic-sense cDNA copy of the three viral segments (Figure 1.11). Here, RNA transcription is usually under the control of a T7 promoter, and T7 RNA polymerase is delivered to the cells either through a helper vaccinia virus (vTF7-3) or fowlpox virus (FPT7) or it is constitutively expressed, for

example, in BSR-T7/5 cells (Buchholz et al., 1999). Support/helper plasmids encoding

viral proteins N and L are also transfected in some cases in order to boost initial transcription (Kawaoka & Neumann, 2004; Bouloy & Flick, 2009). Further, transcription efficiency of the T7 promoter in the transcription plasmid is increased with the addition of a G residue (either one, two or three) immediately after the promoter

sequence. Billecocq et al. (2008) found that infectious RVFV could only be produced

when at least two of the transcription plasmids had at least one ‘G’ after the T7 promoter. The authors found that, without that increased efficiency the number of

produced transcripts were too low to initiate replication (Billecocq et al., 2008). Hence,

bunyavirus transcription plasmids contain one, two or three G’s immediately after the T7 promoter sequence. The newly generated viral RNA transcripts do not function if they contain extra nucleotides at the 3’ UTR, and so to prevent this, a self-cleaving hepatitis delta virus (HDV) ribozyme sequence is usually placed just before the T7

terminator (Figure 1.11) (Perrotta & Been, 1990; Schnell et al., 1994; Bouloy & Flick,

2009). A study by Ghanem et al., (Ghanem et al., 2012) demonstrated the inability of

rabies virus to replicate when the transcripts contained extra nucleotides at their 3’end, whereas 5’ UTR overhangs were tolerated. The authors were able though to enhance rescue efficiency by using a hammerhead ribozyme (HHrz) to generate exact 5’ ends as well.

Although the T7 system is the preferred method to rescue bunyaviruses the cellular polymerase I/II system has also been used. This system was initially developed for viruses that replicate in the nucleus, such as influenza viruses (Kawaoka & Neumann,

2004; Billecocq et al., 2008). In 2001 Flick et al. (Flick & Pettersson, 2001) used the

pol-I derived CCHFV (Flick et al., 2003b) and HTNV (Flick et al., 2003a) minigenome

segments could be packaged. Then in 2007 Ogawa et al. (Ogawa et al., 2007) rescued

AKAV using this system. In 2008, Billecocq et al. (Billecocq et al., 2008) conducted a

comparative study of both transcription systems using RVFV. Results from the study demonstrated that both the pol-I system and the T7 system efficiently generated high titres of infectious virus. Results also demonstrated that it was only the T7 system that produced infectious virus in the absence of the N and L helper plasmids. The N and L transcripts that are generated from the transcription plasmids are sufficient to initiate further steps, and hence bunyavirus rescue systems are successful with three plasmids (Bridgen & Elliott, 1996).

Some bunyaviruses are, however proving to be difficult to rescue, for example viruses

of the Hantavirus genus. Considerable efforts are being made towards understanding

the underlying causes preventing virus recovery in cultured cells. With the current advancement in sequencing technology, comparisons between the dynamics of clinical isolates and cell-culture adapted virus populations will become easier, and with our growing understanding of how bunyaviruses function we may be a step closer to creating viruses from this important genus as well. These viruses do prove that although we have come a long way since Dimitrii Ivanovsky first observed tobacco mosaic “disease” in 1892, we still have a long way to go in our understanding of how a virus population functions.

Minigenome and Virus-like Particle (VLP) production assay

Minigenomes and Virus-like Particle (VLP) assays can also be used to study various aspects of the virus life cycle without the need to rescue infectious virus. This can be especially useful when viruses require high containment for their use, like CCHFV, which requires a biosecurity level 4 laboratory. In the minigenome system viral UTRs flank reporter genes such as Green Fluorescent protein (GFP), CAT or luciferase. These genes are placed in a viral genomic sense (negative-sense), so expression can only be derived with the correct viral L and N protein. Hence, the system also serves as a way to test viral UTRs and protein-coding genes for functionality before attempting to rescue the virus (Figure 1.12). The minigenome system has served as a way to study

bunyavirus transcription, encapsidation and promoter strength (Dunn et al., 1995;

Weber et al., 2001; Blakqori et al., 2003; Flick et al., 2003a; Kohl et al., 2004b;

Ikegami et al., 2005; Bergeron et al., 2010). In 2006 Shi et al. demonstrated that by

including a glycoprotein expression plasmid in the minigenome system VLPs could be

generated, Figure 1.12 (Shi et al., 2006). VLPs are formed because the L and N proteins

interact with the reporter segment to form RNPs, and as the authors subsequently demonstrated using this assay, the RNP interacts with the Gn-CT in order to assemble

and eventually bud out as a virion (Shi et al., 2006; Shi et al., 2007). Overby et al.

(Overby et al., 2006) compared UUKV-VLPs to authentic UUKV-virions showing that

their morphology in cell culture was identical. Since VLPs do not contain all the elements of an authentic virus they are not capable of further rounds of replication.

Recently Devignot et al. demonstrated that by transfecting the “VLP-recipient” cells

with L, M and N expression plasmids VLP production could be increased, which, as the authors point out, could be highly beneficial for VLP-based vaccine/antiviral

Figure 1. 11. Bunyavirus rescue system.

(A) BUNV UTR sequences. Sequences are presented in the antigenomic sense. Nucleotides in red highlight the mismatch at nucleotide number 9. The black line separates the first 11 nucleotides that are conserved for all three segments and within the

Orthobunyavirus genus. Positions 8 and 9 vary in some viruses. The red shading shows

the nucleotides that are conserved for that particular segment in the Orthobunyavirus

genus, nucleotides beyond this vary for each virus. (B) Rescue system based on BUNV. The viral sequences shown in (A) need to be accurate in order for them to function (black box in the transcription plasmid). The transcription plasmid contains a T7 promoter and terminator, a hepatitis delta ribozyme and the cDNA copy of the viral segment. These are transfected into cells that express T7 polymerase to allow transcription from the plasmids. Viral proteins expressed are sufficient to initiate replication. Figure B adapted from (Elliott, 2014).

3’ - T C A T C A C A C G A T G G C T A T T G T T T T G T C G G A | | | | | | | | | | | | | | | | | | | | | | 5’ - A G T A G T G T A C T A C C G A T A C A T C A C A A A C C T 3’ - T C A T C A C A C G A G G A T G T A T T C T T T T A A C A T | | | | | | | | | | | | | | | | | | | | | | | | 5’ - A G T A G T G T A C T C C T A C A T A T A G A A A A T T T A L" 3’ - T C A T C A C A C G A G G T G G A T T T T G A A T T T T A T | | | | | | | | | | | | | | | | | | 5’ - A G T A G T G T A C T C C A C A C T A C A A A C T T G C T A M" S" A B Nucleus' T7'promoter' T7'terminator' 5’' 3’' cDNA'(viral'sequence'in'an:genome'sense)' Hepa::s'delta'ribozyme' Transcrip)on+plasmid:+ T71expressing+cells+ Transfec:on' 3'transcrip:on'plasmids'' (L,'M,'S)' SelfGcleavage' T7'polymerase' Transcrip:on' Transla:on' N' NSs' S+ L' L+ Gn''Gc'''NSm'' M virus'

Figure 1. 12. Schematic of a minigenome and VLP assay.

The diagram explains how a minigenome and a VLP (virus-like particle) assay are carried out. The minigenome plasmid contains a reporter gene flanked by viral UTRs in the genomic sense orientation. Expression plasmids encode the viral L (polymerase) and N (nucleocapsid) protein, and in order to produce VLPs an M (glycoprotein) expression plasmid is used as well. If the plasmids are under the control of a T7 promoter, cells must be able to produce T7 RNA polymerase (eg. BSR-T7/5 cells).

Minigenome assay VLP assay

L T7 N T7 Expression plasmids Minigenome plasmid

Renilla luciferase gene under the control of viral UTRs

co-transfection BSR-T7/5 Renilla Light L, N & UTRs functional Reni lla 5’ 3’

(cells that produce T7 polymerase)

Lyse cells and measure light activity 24/48 h post transfection

+

M

T7

harvest VLP