Tecnologías de Información y Comunicaciones

Attachment and entry

Similar to other enveloped viruses a bunyavirus virion utilises its glycoproteins Gn and Gc to attach and enter into a host cell. The type of cellular receptor the virion binds to will determine the cell tropism of that virus. Current understanding of host cell receptors for bunyaviruses are limited, however knowledge of the varied cell tropism of some bunyaviruses indicate that these viruses may have evolved to interact with a number of mammalian cell receptors (Elliott & Schmaljohn, 2013). Phleboviruses RVFV, SFTSV, TosV, Punta Toro (PTV) and UUKV, and orthobunyavirus Germiston virus were shown to interact with host cell receptor DC-SIGN (DC [dendritic cell] - specific ICAM [intercellular adhesion molecule] - 3 grabbing non-integrin). DC-SIGN is a receptor present on immature DCs, which reside in peripheral tissues and are likely the first cells to encounter incoming viruses. DC-SIGN is a type II membrane protein with a calcium-dependent lectin extracellular domain likely capable of interacting with the glycosylated sites on the viral glycoproteins (Lozach et al., 2011; Hofmann et al., 2013). This interaction would then trigger a response in the DC causing it to mature into an antigen-presenting cell (Tan & O'Neill, 2005). As number of viruses appear to have evolved to use DC-SIGN as entry into mammalian cells they have also evolved mechanisms of blocking the maturation of these cells (Rogers & Heise, 2009). Phleboviruses RVFV and TosV have also been shown to interact with the proteoglycan heparin sulfate receptor (Jin et al., 2002; de Boer et al., 2012b). Whilst, pathogenic hantaviruses can interact with integrins β1, β2 (CR3 and CR4) and β3 (Gavrilovskaya et

al., 1998; Gavrilovskaya et al., 1999; Raftery et al., 2014), as well as Decay- accelerating factor (DAF)/CD55 and gC1qR/p32 (Choi et al., 2008; Krautkramer & Zeier, 2008) for attachment to endothelial and epithelial cells.

Upon attachment bunyaviruses take advantage of the endocytic pathway for internalization. OROV, LACV, HTNV and CCHFV were shown to use the clathrin- mediated endocytotic (CME) pathway (Figure 1.6 A) (Jin et al., 2002; Santos et al., 2008; Simon et al., 2009; Hollidge et al., 2012). CME is used by all cells and is probably the reason why several enveloped viruses use it to gain entry into cells (Figure

1.5 A, step 2). Interestingly, UUKV appears to predominantly use a clathrin- independent pathway to enter A549 (human) and BSC-40 (money) cell-lines (Lozach et al., 2010). Akabane virus (AKV) on the other hand uses the clathrin-independent pathway in bovine-derived cell-lines, whereas in non-bovine cells it seems to use the CME pathway (Bangphoomi et al., 2014).

Membrane fusion

The endocytic pathway follows a pH gradient that can be detected by bunyavirus glycoproteins triggering a conformational change in their structure (Överby et al., 2008). This pH sensing is possible due to protonation of the histidine residues typically found on the viral fusion protein (Kampmann et al., 2006; Mueller et al., 2008). As discussed previously, Gc likely functions as a fusion protein. This is further strengthened by work on RVFV, where certain conserved histidine residues on Gc were found to be important for virus infectivity (de Boer et al., 2012a). Conformational changes in Gc would then mediate fusion between viral and endosomal membranes, hence allowing release of viral RNP into the cell cytoplasm (Figure 1.5 A, step 3) (Mercer et al., 2010; Cosset & Lavillette, 2011). Several enveloped viruses are known to use this mode of penetration (White et al., 1981; Kielian et al., 2010). Detailed work by Lozach et al. demonstrated that UUKV is transported from the early endosomes (pH <6.3) to late endosomes (pH <5.3) before infection occurs (Figure 1.6 B). The authors demonstrated that infection was pH dependent since neutralisation of vesicular pH inhibited infection, whilst acidification of the external cell environment was sufficient to trigger fusion of the viral and cell plasma membranes (Lozach et al., 2010). Cell-to- cell fusion has been shown using different bunyaviruses, where over-expression as well as infection induces syncytium formation (Jacoby et al., 1993; Hacker & Hardy, 1997; Ogino et al., 2004; Plassmeyer et al., 2005; Shi et al., 2007).

Transcription and Replication

Bunyaviruses replicate in the host cell cytoplasm and progeny virions mature and bud at the Golgi apparatus, Figure 1.5 A (Shi et al., 2010; Elliott & Schmaljohn, 2013). Upon release of RNP the virion-associated L begins transcription of the genome via a cap- snatching mechanism (Figure 1.5 B). Work on SNV suggests that N can recognise 5’

this using BUNV and RVFV demonstrated that both L and N are together required for active transcription (Dunn et al., 1995; Lopez et al., 1995). As N also encapsidates the genome, structural data suggests that N could potentially expose the UTRs using its flexible arms allowing L to bind (Elliott, 2014). Recent structural data on LACV L bound to vRNA revealed that the terminal 3’ and 5’ UTR sequences are crucial and that they each bind specific regions in the L molecule, confirming all prior in vitro

mutagenesis work. The overall crystal structure of L indicates a main globular core, which harbours the RdRp and RNA-binding domains connected to a flexible endonuclease domain by a linker region. The template entry and exit tunnels and the nascent RNA exit tunnels are located in the main globular core (Gerlach et al., 2015). Transcription occurs prior to replication and unlike transcription, replication occurs in a cap-independent manner. The exact mechanism for switching from cap-dependent to cap-independent initiation is still uncertain, but the possibilities of host cell translation shut-off forcing this switch; or that viral and/or host proteins may be involved in the process have been proposed (Guu et al., 2012; Elliott & Schmaljohn, 2013). In transcription the nascent mRNA terminates upstream of the 5’ end, however in replication the nascent strand is processed right to the very end of the 5’ termini. Transcription termination signals ubiquitous to all three segments have not been found, but a pentanucleotide sequence 5’-UGUCG-3’ in BUNV S segment appears to be able to signal termination (Barr et al., 2006; Ikegami et al., 2007; Blakqori et al., 2012). Translation commences immediately on the growing nascent mRNA strand and is required to prevent this strand from hybridising to the template and halting transcription. These mRNA species are not poly(A) tailed. The L and S segment mRNAs are translated by free ribosomes, whilst M is translated by membrane-bound ribosomes (Elliott & Schmaljohn, 2013; Elliott, 2014). Once in the replication mode it is suggested that nascent cRNA undergoes immediate circularisation by polymerase dimerisation followed by immediate N encapsidation. This replicative intermediate RNP is known as the antigenome (Figure 1.5.B) and it serves as a template for another round of replication (Gerlach et al., 2015).

Release

The M segment products Gn and Gc are post-translationally modified by N-linked glycosylation. Primary glycosylation takes place at the ER (Elliott & Schmaljohn, 2013; Elliott, 2014). All viral proteins then migrate towards the Golgi and signals in the Gn cytoplasmic tail (CT) help recruit RNP for assembly (Shi et al., 2007; Strandin et al., 2013). Early EM work on UUKV and BUNV demonstrated viral factories at the Golgi complex where these viruses appear to bud (Figure 1.6 B and C) (Kuismanen et al., 1982; Salanueva et al., 2003; Fontana et al., 2008). Mature bunyaviruses are then transported to the plasma membrane in large vesicles via the secretary pathway (Figure 1.6 D) where membrane fusion then enables the virions to exit the host cell (Figure 1.5 A, step 8-10; Figure 1.6 D and E) (Elliott & Schmaljohn, 2013; Elliott, 2014).

Figure 1. 5. Life cycle of the bunyaviruses.

(A) Schematic diagram of the various stages in a bunyavirus life-cycle. EE, early endosomes; LE, late endosomes; ER, endoplasmic reticulum; SV, secretary vesicles. (B) Transcription and replication of the bunyavirus genome. The genome is in a negative-sense orientation and is transcribed into a replicative intermediate known as the antigenome. Red circles depict the N protein encapsidating the genome/antigenome. The red dashed-box highlights that the mRNA contains 10 – 15 nt long host-derived primers/caps. Numbers

5’ 3’ 3’ 5’ 6. Genome replication 5’ 3’ host-derived cap 5. Translation 4. Transcription Protein RNA species Antigenome (+) Genome (-) mRNA (+) bunyavirus Nucleus ER Golgi L S M L S M L = S = M = L S M L S M L S M L S M L S M

1. Attachment to cell receptor

L S M SV L S M L S M L S M 2. Endocytosis L S M 3. Fusion EE LE Lysosome 4. Transcription 5. Translation 6. Genome replication 7. Assembly 8. Migration 9. Fusion 10. Egress 5’# 3’# 3’# 5’# 5’ 3’ 5’# 3’# 3’# 5’#

A

B

Figure 1. 6. Electron micrographs of bunyavirus entry and exit from the host cell. (A) OROV (arrowhead) entering HeLa cells via clathrin-coated pits (arrow). Image

taken from (Santos et al., 2008). (B) UUKV inside early (EE#2) and late (LE)

endosomal vesicles in A549 cells. Image taken from (Lozach et al., 2010). (C) Viral

factories of BUNV in the BHK-21 cells. G, Golgi; V, virus particle. The arrows show various tubular and globular structures that form part of the viral factories. Image taken

from (Fontana et al., 2008). (D) BUNV inside the Golgi in BHK-21 cells (arrows), post-

Golgi area (arrowhead). (E) BUNV exiting BHK-21 cells by secretary vesicles (SV) (F). Exited BUNV attached to the cell surface. Images (D), (E) and (F) were taken from

(Salanueva et al., 2003), where the cryosections were labelled with anti-Bunyamwera

A B C D E F 150 nm 100 nm 100 nm 100 nm 100 nm 100 nm