STRUCTURAL AND FUNCTIONAL INSIGHTS INTO THE REPLICASE/TRANSCRIPTASE SYSTEMS OF THE DEADLY BAT-BORNE HUMAN VIRUSES: MARBURG, EBOLA, AND NIPAH

Replication and transcription of genomes in the human pathogenic viruses, viz. Marburg, Ebola and Nipah are accomplished by a virally encoded RNA-dependent RNA polymerase (RdRp). The enzyme performs this dual function with a polymerase cofactor, VP35, in Marburg and Ebola viruses and a phosphoprotein, P, in Nipah and its related viruses. As RdRp is indispensable for the viral lifecycle, it is one of the most attractive molecular targets for the development of broad-spectrum antiviral therapeutics to control the spread of these viruses. However, until now, little is known about the RdRp of these viruses, especially with respect to their active sites, polymerase catalytic centre and its catalytic mechanism, which are considered the most promising molecular targets for drug development initiatives. In this study, multiple sequence alignment (MSA) was integrated with structural information from X-ray crystallography, cryo-electron microscopy (cryo-EM) and site-directed mutagenesis (SDM) studies to identify the putative catalytic and metal-binding sites of the RdRp from Marburg, Ebola and Nipah viruses. MSA analysis suggests that the RdRp from these viruses uses an -LA/VG- as the template-binding pair, an invariant K, as the proton abstractor which initiates the catalysis and an invariant R at -4 from the catalytic K, as the nucleotide discriminator, all of which are highly or completely conserved.  Besides, the Marburg and Ebola viruses contain an additional second putative polymerase catalytic core within the N-terminal domain (NTD), which is absent in the Nipah virus polymerase. The identified catalytic residues exhibit strong structural similarity to the conserved polymerase catalytic site architectures already reported in numerous RNA- and DNA-dependent polymerases, supporting a common catalytic framework. The catalytic magnesium-binding sites are also completely conserved among these viruses and consist of two invariant aspartate-containing motifs, -GDN- and -TDL-. This arrangement differs slightly from that what is reported for several other non-segmented negative-sense RNA viruses, including influenza, rabies, mumps and measles, which typically use -GDN- and -G/SDD- motifs for catalytic magnesium-binding. Furthermore, the RdRp domains of all three viruses contain two putative zinc-binding motifs (ZBMs), suggesting an important structural role in polymerase stability and function. Interestingly, the RdRp of Marburg and Ebola viruses, members of the Filoviridae family, possess an additional putative HNH-endonuclease motif(s) that is absent in the Nipah virus polymerase, a member of the Paramyxoviridae family. Collectively, these findings reveal conserved structural and catalytic features of the polymerases from these bat-borne human pathogens, and provide new insights into their evolutionary relationships, and also identify potential molecular targets for rational antiviral drug design.