Phleboviruses replicate in a seven-step process. First, the cellular attachment is driven through the
glycoprotein interactions with host cells. Examples of this are Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Non-Integrin (
DC-SIGN),
heparan sulfate (HS), or Non-Muscle Myosin Heavy Chain (NMMHC-IIA). Second, in the late
endosome, the low
pH causes fusion activity in the membrane of the Gc protein. Uukuniemi virus (UUKV) penetration is promoted by the expression of
vesicle-associated membrane protein 3 (VAMP3). Additionally, the fusion of
Rift Valley fever virus (RVFV) in late endosomes is inhibited by the
interferon-induced transmembrane proteins 2 and 3 (IFITIM2 and
IFITIM3). Third, the viral and endosomal membranes are fused to allow for the release of the viral
ribonucleoprotein complexes into the
cytoplasm (Also the site of viral
transcription and
replication). Fourth, the
precursor protein, Gn/Gc, is translated at the
rough endoplasmic reticulum (ER). This precursor protein is cleaved by signal
peptidase. Synthesis of the viral nucleoprotein and viral
polymerase in the cytoplasm combines with the newly formed genomic RNA (gRNA) ribonucleic protein complexes (RNP). Fifth, two ER chaperones,
binding immunoglobulin protein (BiP) and
calnexin, are required to ensure proper folding of GN/Gc. Gn/Gc are similarly catalyzed by
protein-disulfide-isomerase through the formation of
disulfide bonds. At the same time,
calreticulin prevents any
misfolded Gn/Gc from being exported to the Golgi. Sixth, The correctly folded Gn/Gc
heterodimers are transported to the
Golgi apparatus. The cytoplasmic tails of Gn in the budding process associate with RNPs during this time. Seventh, once the budding of the new virus particles is completed,
vesicles that contain the virus are transported to the
plasma membrane to be released by
exocytosis.
Role of Gn and Gc in phlebovirus entry A study of the family
Bunyaviridae showed that bunyavirus particles are
pleomorphic. This known fact caused some surprise when studies showed that UUKKV and RVFV particles are spherically shaped and highly ordered. The configuration of Gn and Gc proteins in the
viral envelope imposes the order of the particle. The viral envelope forms an
icosahedral lattice with a triangulation number of 12. Also included in the lattice composition are 110 hexametric and 12 pentameric
capsomeres. For RVFV in particular, 720 Gn/Gc heterodimers are included in the capsomeres. In these cases, Gn forms the spikes of the capsomere while Gc is closer to the
lipid membrane, thus placing it underneath. The pH surrounding the capsomere ultimately determines its shape. This is largely due to protonation triggering conformational changes in Gc, commonly included with membrane fusion. An assembly model for the RVFV envelope has been proposed which consists of Gc dimers positioned horizontally with respect to the viral membrane. This is known because the RVFV Gc ectodomain is crystallized as a dimer. This is opposed to the virion interior of bunyaviruses which has no matrix protein, and thus, has no defining organization. This means that on the virion surface, the Gc and Gn proteins must be present in a highly ordered placement. To begin entry, phlebovirus particles bind to various components of the plasma membrane. These components interact with the viral glycoproteins of phlebovirus and regulate entry efficiency. While these components are not crucial to the actual entry of the virus itself, receptors are components of the plasma membrane that bind to the glycoproteins and are critical for entry. In phleboviruses, it was determined that glycan-protein interactions play a crucial role in phlebovirus entry. Heparan sulfate (HS) is another crucial component aspect in phlebovirus attachment. It is a
glycosaminoglycan (GAG), which is an unbranched
polysaccharide made of
disaccharide repeats, that results in the creation of a
proteoglycan. Cell lines with defined glycosylation defects were analyzed and showed that HS is necessary for the entry of RVFV. This was confirmed by the removal of HS using an enzyme. HS is charge-dependent in their interactions with virus particles, and studies showed that there are basic amino acid clusters on the P78 protein that interact with negative sulfate clusters on HS. In comparison, there were no identified HS binding sites on Gn and Gc. The P78 protein is plentiful in RVFV-infected cells in insects, while infected cells in mammals produce significantly less P78 proteins. The P78 protein is much more efficiently produced in RVFV in mosquito cells as it is required for viral spread in mosquitos. Overall, the cell line is heavily dependent on HS in RVFV entry. A computational study provided evidence that phlebovirus Gc proteins might be class II
membrane fusion proteins. Final proof for this theory was given by the elucidation of the
ectodomain's structure of RVFV Gc in its pre-fusion status. Gc has three domains with characteristics that resembled other class II proteins. An internal fusion loop was discovered, which is a critical aspect of all class II proteins. The location of a
histidine in Gc resembled a pH sensing feature, which matches class II characteristics. Although there were many similarities within the structure of the RVFV Gc and class II proteins, the interface between domains I and II in RVFV Gc is more rigid and bigger than other class II proteins. Additionally, RVFV Gc has more disulfide bridges centralized in different locations than the other compared proteins. However, its overall structure and functionality is most closely resembling a class II membrane fusion protein compared to any other class. The pH sensing feature in the Gc protein is of note. The membrane fusion activity of the phlebovirus Gc proteins is very dependent on pH, as a low pH triggers the transport of virions into
endolysosomes. Elevating intravesicular pH inhibits phlebovirus entry. However, it is still unclear whether Gc proteins must bind to a receptor before being triggered by pH or not. After the viral and endosomal membranes have been fused together, the L,M, and S genomic segments (associated with viral polymerase) are released into the cytoplasm. This initiates the transcription to genomic RNA into
mRNA. Viral proteins begin undergoing
translation before the transcription of mRNA has finished. The Gn and Gc phlebovirus proteins are encoded on the M-segment and undergo
synthesis. The precursor Gn/Gc protein cannot be detected in a cell already infected with phlebovirus. It only is visible after the expression of the M-segment. If
microsomal membranes are present, the precursor is cleaved, indicating cleaving by a host factor during the synthesis of the viral protein. The signal peptidase complex in the ER membrane is responsible for the cleaving of the precursor. This precursor is then translocated into the ER lumen, in which two
hydrophobic domains are inserted with a third, cleaved hydrophobic domain in between. == Emerging group of arthropod-transmitted pathogens ==