*7.2. F protein*

The F proteins observed in SSPE cases present several mutations conferring a hyperfusogenic phenotype. F is produced as metastable protein in its pre-fusion state. This pre-fusion state is generally less stable in the CNS isolates. The F can also fuse without H engagement to any known receptor. Thus, it is suggested that these mutations facilitate CNS spread [40].

Mutations can occur in the HRC domain (T461I, A440P, N462S, N465S, and L454W), in the HRN domain (G168R/E170G), in between HRC and HRN domains (S262G), in the cytoplasmic tail domain (CTD) (R520C, L550P), and in the F2 subunit of F protein. Among the mutations found in the F-SSPE sequence from South African patient (G168R/E170G/S262G/A440P/R520C/L550P and X551G), only the mutation S262G (position already associated to hyperfusogenicity with a mutation S262R) located at the interface of three protomers, involved in fusion activation, may independently confer an hyperfusogenic phenotype to F without needing any other mutation. The functional analysis of MeV\_IC323 virus carrying this F-SSPE with all seven mutations confirmed the finding that an SSPE strain can disseminate via cell-to-cell spreading in Vero cells, in the absence of known receptors [40] (Figure 3B).

The mutation of stop codon (X551G) in F-SSPE strains has been frequently observed previously [107] and leads to an elongated cytoplasmic tail (called LT for Long Tail) that can enhance the incorporation of F and nonspecific cellular protein in the virion [121,122].

Other mutations found in F extracellular domain from SSPE sequences isolated from patients brain (T461I and S103I/N462S/N465S) also confer hyperfusogenicity and can spread in human neuroblastoma cell lines and suckling hamster brains in the absence of known MeV receptors [123,124].

Fusion inhibitors such as 3G or FIP are tested on MeV and it has been documented that several mutations emerged in F protein in order to escape the treatment. The impact of these mutations (I87T, M94V, S262R, L354M, A367T, N462K) on the fusion machinery is of great interest [125–127]. One of the most interesting mutations that emerged is located at the residue 262. The escape mutation S262R confers hyperfusogenicity, as well as the mutant S262G that has been described in a real case of SSPE [40,123]. These data highlight the fact that emergence of mutations under a selective pressure can lead to viral adaptation to CNS. This can also allow a better design of inhibitors that could counteract these adaptation mutations.

As discussed in Section 6.2. the hyperfusogenicity correlates with a lower thermal stability of the pre-fusion state of F [28,125]. As an example, the L454W F is highly unstable and this characteristic could be sufficient to trigger F in a postfusion state by itself, allowing the fusion to occur without any receptor engagement. In the context of a circulating viral particle outside the brain, that property might not be an advantage for the virus, which could explain why any hyperfusogenic form of MeV has never been found in circulating viruses.

#### *7.3. H Protein*

The H protein of SSPE strains is often mutated as well and contributes to neurovirulence [128]. In a recent study, three mutations were found in the H gene of South African SSPE strain, in the cytoplasmic tail, the stalk domain, and β5 blade of the head domain, associated with substitutions R7Q, R62Q, and D530E, respectively [40]. The residue D530 is necessary for cell entry through SLAM, so the mutation D530E could compromise the use of infection through SLAM [129,130].

In a modified Edmonston strain expressing a murine-adapted H protein from a neurovirulent strain CAM/RB, the substitutions G195R and S200N lead to complete loss of neurovirulence in mice C57BL/B6 [83,131,132]. Due to questionable strains and animal model used in this study, these data have to be considered carefully and these findings might be difficult to transpose to human SSPE cases. Nevertheless, it highlights the potential existence of a specific site in H involved in neurovirulence or a site of an unknown neuron-specific receptor.

C-terminus elongation of the H protein due to single-point mutation at the stop codon have also been reported multiple times in SSPE cases [75,133]. Contrarily to deletions of the cytoplasmic tail of H which were shown to enhance fusion activity [121], elongation of the extracellular domain of H seemed to impact binding, targeting, and may explain at least partially the high level of antibodies in SSPE cases [75,133].

Unlike SSPE, mutations in H gene of MIBE virus sequences seem to be less frequent and further investigations for their potential impact in CNS infection is required [92].

## *7.4. Mutations in Other Genes*

In SSPE cases, some mutations have also been found in N, P, and L proteins but most of the recent studies focused on F and M proteins. Some P genes from SSPE cases exhibit an impaired editing system that lead to less V protein production. Most of the time, the viral cycle does not seem compromised but the lower expression level of V could contribute to the viral persistence by reduced inhibition of interferon (IFN) response [134]. Is has also been shown that the P gene of the multi-mutated rodent brain-adapted strain CAMR40 is largely involved in neurovirulence, suggesting that MeV P gene could also play a role in CNS infection [135].
