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protein successfully replicate in vitro and in vivo (Biacchesi et al., fusion the ribonucleoprotein complex (RNP) made up of the N,
2004b; Naylor et al., 2004) showing that F has both attachment P and L proteins containing the viral RNA (vRNA) is released
and fusion properties. It has been suggested for human Metap- into the cell cytoplasm for transcription of mRNA or to serve
neumoviruses that the interaction of F with target cells involves as template for complementary RNA (cRNA) and subsequent
the integrin binding domain RGD with an integrin αvβ1 receptor. vRNA production. After the synthesis of vRNA and proteins,
It is unknown if similar interactions occur for the avian viruses the M protein and RNPs are transported directly to the cell
however, no RGD binding domain has been identified in the F membrane. The glycoproteins F, SH and G pass through the
proteins of AMPVs. The F protein is also highly immunogenic and endoplasmic reticulum (ER) to the Golgi apparatus and then to
contains important epitopes for protective immune responses the plasma membrane. Here all the components are assembled,
(Brown et al., 2009; Hu et al., 2017). and new virions are released by a budding process.
Of all the viral proteins, the functional role of SH remains the A recent study looking at the late stages of the HMPV
most elusive. Several groups have demonstrated that it is dispen- virus replication cycle showed that not all subsequent
sable for AMPV, HMPV and RSV viability though its deletion infections result from the budding/attachment mechanism
does result in some level of attenuation. SH-deleted AMPVs also described above. In this study a new role for the P protein
have an altered cytopathic effect in vitro (Ling et al., 1992; Naylor was observed as it interacted with the actin cytoskeleton in
et al., 2004). Recently the SH protein of HMPV has been shown human bronchia airway cells leading to extensive branched
to inhibit and down-regulate certain processes in different cellular networks between cells where viruses and or just RNPs were
signalling pathways (Bao et al., 2008a; Hastings et al., 2016). It has transmitted (Fig. 4.4) without ever passing via the extra cel-
also been recently reported that like the SH of RSV, the HMPV lular environment (El Najjar et al., 2016). As cited in that
SH forms oligomeric structures in the target cell membrane paper cell-to-cell spread independent of particle release has
making the membrane more permeable (Masante et al., 2014). also been reported for the closely related RSV, measles virus,
For AMPV more studies are required concerning the functional influenza A virus and parainfluenza virus 5 (Shigeta et al.,
roles of this small hydrophobic protein. 1968; McQuaid et al., 1998; Lawrence et al., 2000; Makhor-
Finally, the surface G protein, which is the most variable MPV tova et al., 2007; Roberts et al., 2015; Singh et al., 2015). A
protein in terms of amino acid sequence, is considered responsi- mini review dedicated to these mechanisms has also recently
ble for virus attachment to cells however; as discussed above it is been published (Mothes et al., 2010).
now clear that it is not the only protein capable of carrying out
this process. Similar to the F protein it is highly immunogenic and
contains important epitopes for protective immune responses Reverse genetics
(Hu et al., 2017; Naylor et al., 2007). Similar to the SH protein ‘Reverse genetics’, as the term implies, is the opposite of ‘regular
it is also emerging as an inhibitor of cellular immune responses genetics’. In brief, regular genetics looks at the genetic basis of
(Bao et al., 2008b, 2013; Kolli et al., 2011). a phenotype whereas reverse genetics looks at phenotypes that
arise from specific changes introduced into genetic sequences. In
virology, reverse genetics systems allow the rescue of infectious
Viral replication viruses that carry precise genome sequences from cloned cDNA
Most of the current understanding for the AMPV replication so as to study their phenotypic effect.
cycle is based on that of other negative-sense single-stranded The first reverse genetics (RG) system that allowed the rescue
RNA viruses, above all HRSV. AMPV replication takes place in of infectious AMPV from cloned cDNA was reported in 2004
the cytoplasm of infected cells. Firstly, the virus attaches to the (Naylor et al., 2004). In the same year RG systems were developed
surface of target cells, the process of which is mainly directed for HMPV (Biacchesi et al., 2004a; Herfst et al., 2004). RG sys-
by the surface glycoprotein G, for RSV this involves binding to tems were then subsequently developed for AMPV-C (US turkey
glycosaminoglycans especially heparin sulphate and chondroitin strain) (Govindarajan et al., 2006), AMPV-B (Laconi et al., 2016)
sulfate B (Collins and Karron, 2013) however it is now clear and most recently for AMPV-C (EU duck strain) (Szerman et al.,
that the fusion protein also plays a part in this process (Ling et 2018). Although varied methods were used to construct these
al., 2008; Cseke et al., 2009; Wei et al., 2014). After attachment systems, all are based on the co-transfection of plasmids encoding
of MPVs to the target cell, the F protein promotes fusion of the the proteins of the RNP complex plus M2 and the viral genome
viral envelope and the plasma membrane. The factor triggering either in the negative or positive sense. Interestingly all these
fusion remains unclear though it has been suggested, at least for systems have been placed under the control of a T7 promoter
HMPV, that it could be the result of the binding to integrin αvβ1 sequence for which the T7 polymerase has either been delivered
(Schildgen et al., 2011). This seems unlikely for AMPVs as the using helper viruses or by cell lines that are maintained to consti-
conserved αvβ1 integrin binding motif of RGD of the F protein tutively express it by antibiotic selection. None have been based
of HMPVs (Cox et al., 2012) is not present in any of the known on the eukaryotic pCMV promoter which would seem an easier
AMPV F protein sequences. These have been reported to be RSD option as it would eliminate the need for helper viruses and put
for all AMPV C viruses and RDD for AMPV-A, B and D (Brown less restriction on the cell type required for rescue. Furthermore,
et al., 2014). a dual plasmid system with one plasmid encoding the genome
As previously reviewed (Schildgen et al., 2011), following and the other all four support proteins, similar to that described