The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and

A Look into Bunyavirales Genomes: Functions of Non-Structural (NS) Proteins

“…However, the EBOV MLD is a domain of the structural glycoprotein [ 58 ] and likely unique from the CCHFV MLD [ 48 ]. In a study of CCHFV transcriptionally competent virus-like particle (tc-VLP) replication, deletion of MLD had no impact on particle infectivity although it reduced incorporation of glycoproteins into particles by about 60%, while the MLD-GP38 double deletion inhibited assembly of infectious tc-VLPs [ 59 ]. GP38, encoded between the MLD and pre-Gn of the M polyprotein ( Figure 3 ), is generated by host proteases using highly conserved furin and SKI-1 cleavage sites [ 48 ].…”

“…However, CCHFV mutants lacking this furin site, and thereby lacking optimal GP38 release, have only slightly decreased Gn maturation and transient reduction in virus titers, indicating that the furin site is not required for viral replication, and the reduction in viral titers may be due to either or both of decreased GP38 and mature Gn production [ 44 ]. In the same tc-VLP experiment, loss of infectivity from MLD-GP38 double deletion was associated with impaired Gc maturation, showing a dual effect of GP38 on both Gn and Gc trafficking to the Golgi, where both proteins are processed [ 44 , 59 ]. This indicates that the MLD may have conformational effects on GP38 that impact its ability to traffic Gc to the Golgi or, MLD may regulate Gc accumulation in the Golgi [ 59 ].…”

“…In the same tc-VLP experiment, loss of infectivity from MLD-GP38 double deletion was associated with impaired Gc maturation, showing a dual effect of GP38 on both Gn and Gc trafficking to the Golgi, where both proteins are processed [ 44 , 59 ]. This indicates that the MLD may have conformational effects on GP38 that impact its ability to traffic Gc to the Golgi or, MLD may regulate Gc accumulation in the Golgi [ 59 ]. Interestingly, targeting of the GP38 by host antibody responses may be protective as mice treated with a non-neutralizing monoclonal antibody recognizing GP38 were protected against lethal disease [ 56 , 60 ].…”

“…The double membrane spanning NSm was identified in 2007 and is found in CCHFV, DUGV, NSDV, and HAZV [ 47 , 59 , 61 ]. NSm is released in the endoplasmic reticulum (ER) where, for CCHFV, the glycoprotein precursor encoded by the genomic M segment is cleaved by subtilase-like proteases into NSm, preGn, and preGc ( Figure 3 ) [ 62 ].…”

“…NSm is released in the endoplasmic reticulum (ER) where, for CCHFV, the glycoprotein precursor encoded by the genomic M segment is cleaved by subtilase-like proteases into NSm, preGn, and preGc ( Figure 3 ) [ 62 ]. In tc-VLP experiments, NSm deletion caused improper Gc processing, defective particle formation, and impaired secretion, although initial trafficking of Gc was unaffected [ 48 , 59 ]. Interestingly, another study, using recombinant CCHFV with NSm deletion, observed that NSm is not essential for viral replication in vitro, as viral growth was only mildly slower compared to WT virus [ 18 ].…”