The first step of the viral replication cycle is the entry process that comprises the attachment and binding of viruses to their host cells and the release of the viral genome into the cytoplasm.
In the case of enveloped viruses, a fusion between the membranes of the virus particle and the target cell has to occur to release the genetic information into the host cell.
The entry of paramyxoviruses is mediated by their surface glycoproteins one of whom is responsible for the attachment and the other for the fusion process.
1.2.1 Viral attachment
Attachment glycoproteins of paramyxoviruses are type II membrane proteins that are integrated into the viral envelope as tetramers consisting of two dimers. Each monomer is composed of an N-terminal cytoplasmic domain (CD), followed by a transmembrane domain
Depending on their binding and enzymatic activity, the attachment glycoproteins of paramyxoviruses are divided into three classes: HN proteins which are expressed by avula- and rubulaviruses attach to sialic acids and are therefore also able to catalyze the cleavage of these neuramic acid derivates to avoid self-aggregation and to enable the release of new virions (Villar et al. 2006). Besides this neuraminidase activity, HN proteins are able to hemagglutinate red blood cells. The H protein which is expressed by morbilliviruses lacks the neuraminidase activity and binds to specific protein receptors e.g. SLAMs or CD46 (Masse et al. 2004; Seki et al. 2020). The glycoprotein G is expressed by henipaviruses, binds to specific receptors such as Ephrin-B2/-B3 and lacks both, neuraminidase and hemagglutination activity (Xu et al. 2012a; Johnson et al. 2015).
Besides binding to cellular receptors, the attachment glycoproteins are further involved in activating the F proteins for membrane fusion. The interaction between the viral glycoproteins is highly specific and occurs only between closely related viruses. According to the “stalk exposure model” of Bose et al., the receptor binding of the attachment protein leads to rearrangements within the head domain, causing a disturbed connection of head and stalk which in turn exposes an F-interaction site in the stalk domain (Bose et al. 2011; Bose et al.
2014).
The HN protein of MuVs has a molecular weight of 74 - 80 kDa and a size of 582 amino acids (aa) (Mahon 2003). HN is the main target of neutralizing antibodies that bind to the head domain where they disturb the binding of HN to cellular receptors and its enzymatic activity (Orvell et al. 1997a; Mahon 2003). It has been shown that MuV HN proteins bind to α2,3-, α2,6- and α2,8-linked sialic acids with different preference depending on the MuV strain (Brostrom et al. 1971; Leprat et al. 1979; Hosaka et al. 1998; Santos-Lopez et al. 2009; Kubota et al.
2016; Kubota et al. 2019).
1.2.2 Virus-induced fusion
The fusion glycoprotein F of paramyxoviruses is a type I membrane protein which is integrated in the viral envelope as a homotrimer. The F protein consists of an N-terminal ED which includes the hydrophobic fusion peptide (FP) and three heptad repeat regions (HRA, HRB, HRC) followed by the TD and the CD at the C-terminal end (Fig. 3). The F protein plays an important role in the viral fusion process by promoting the fusion between viral and host cell membranes (Lamb 1993; Samal 2011). Further, F mediates cell-to-cell fusions: infected cells fuse with neighbored (uninfected) cells and as a result, giant multinucleated cells, so called syncytia, are formed (Wolinsky et al. 1978; McCarthy et al. 1980). Viral spread by release of virions as well as by syncytium formation is therefore possible.
F is synthesized as the fusion-inactive precursor F0 that has to undergo proteolysis and conformational changes to obtain the ability to mediate fusion. The proteolytic cleavage is performed by cellular proteases that recognize a specific cleavage motif directly upstream of the FP. The cleavage motif and thereby also the cellular protease responsible for F cleavage differ among the paramyxovirus species. Many paramyxovirus F proteins such as these of MuV, PIV5 or MeV are processed intracellularly by furin at a multibasic cleavage site within the trans-Golgi network (TGN) (Molloy et al. 1999; Thomas 2002). In contrast to this, F0
precursors of Sendai virus (SeV) or human parainfluenza virus 1 (HPIV1) are first integrated into newly synthesized viral particles before cleavage at a monobasic cleavage site occurs extracellularly by proteases such as transmembrane protease serine subtype II (TMPRSS2) or secreted trypsin-like proteases expressed in the respiratory tract (Hidaka et al. 1984; Kido et al. 1992; Ambrose et al. 1995). In case of henipaviruses, the cleavage involves clathrin-mediated endocytosis of F0 that is first transported to the cell surface. Within the endosome, the cysteine protease cathepsin L (or B) cleaves after a single basic aa residue of F requiring a low pH (Diederich et al. 2005). In the following, the cleavage of paramyxovirus F is described in more detail. The proteolysis results in the cleavage of F0 into the disulfide-linked F1 and F2
subunits (Fig. 3). As a consequence, the FP is present at the new N-terminal end of the F1 subunit to be exposed towards the host cell membrane (Lamb et al. 2006).
irreversible conformational changes at the end of which a six-helix bundle of its HRs is formed (Zhu et al. 2003; Markosyan et al. 2009; Smith et al. 2009). In the pre-hairpin intermediate conformation, the FP of F is exposed and able to contact the host cell membrane: HRA converts into an unstable helical trimeric coiled coil and extends towards the target membrane in which FP is integrated while HRB, also present as coiled α-helices, remains anchored in the viral membrane (Fig. 4). Rearrangements continue as HRB rotates about 180° to bring HRA and HRB into closer contact. A “zipping process” (principle of zipper) follows in which HRA and HRB are anti-parallel orientated and gradually merge, the six-helix bundle starts to be formed and the outer membranes start to fuse resulting in a hemifusion. Next, the inner membranes fuse as well and with the fully completed formation of a stable six-helix bundle structure of F the post-fusion state is reached. Finally, an expanding fusion pore is formed through which the viral RNA genome is released into the cytoplasm. Probably, the actin of the cytoskeleton helps to expand the fusion pore (Smith et al. 2009; Aguilar et al. 2016; Azarm et al. 2020).
Fig. 4: Membrane fusion process mediated by paramyxoviruses. Attachment protein (yellow: head, purple: stalk), fusion protein F (red: fusion peptide FP, blue: HRA and stalk including HRB, green: transmembrane domain, orange: cytoplasmic domain), HRA/B: heptad repeat region A/B. Modified after Aguilar et al., 2016.
1.2.3 Viral replication
The viral replication process of paramyxoviruses takes place in the host cell cytoplasm and is mediated by the RNA transcriptase complex consisting of N, P and L proteins (Fig. 5). Once the viral genome has been released into the cytoplasm of the host cell, the negative-sensed RNA genome serves as template for the RdRp during the transcription as well as the replication process (Lamb et al. 2001). The transcription results in the synthesis of positive-stranded messenger RNAs (mRNA) which are further translated by host cell enzymes. After protein synthesis, the viral proteins are transported to the surface of the infected cell to assemble with the replicated negative-sensed copy RNAs. During their transport to the cell surface, viral glycoproteins are further modified in cellular compartments. These modifications include e.g.
the addition of carbohydrates to and the folding of the viral proteins in the endoplasmic
reticulum (ER) and Golgi apparatus (Watanabe et al. 2019). Also the proteolytic activation of the F proteins by cellular proteases occurs during the cellular transport within different compartments, depending on the virus species and in particular on the proteolytic cleavage motif. Regarding the replication, transcription of the viral RNA genome into a full-length positive-stranded anti-genome template is necessary. The anti-genome is further used by the RdRp to synthesize full-length copies of the negative-sensed RNA genome (Pickar et al. 2014).
Finally, the M protein-mediated assembly of newly synthesized viral proteins and RNA has to take place to form new infectious viral particles. The budding process is the last step of the viral replication cycle and describes the release of newly synthesized virions from the host cell membrane. For those paramyxoviruses, that are capable of binding to sialic acids present on the cellular membrane, the neuraminidase activity of the HN proteins is required to disrupt the binding of virions to the producer cell and to avoid self-aggregation (Takimoto et al. 2004;
Harrison et al. 2010; Aguilar et al. 2011).