The crystal structure of the Hazara virus nucleocapsid protein

Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NP core , both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NP core C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NP core remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus.IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously with self-assembly oligomerization. Here we report the structures of MARV NP core in two different forms. In the MARV NP apo form, we identify an interesting hexamer formed by hydrophobic interaction within a long helix, which is highly conserved and flexible among filoviruses and may indicate its critical function during the virus RNP formation. Moreover, the structural comparison with the NP-VP35 peptide complex reveals a structural transition chaperoned by VP35, in which the RNA binding groove undergoes a transition from closed state to open state. Finally, we discussed the high conservation and critical role of the VP35 binding pocket and its potential use for therapeutic development.KEYWORDS Marburg virus, nucleoprotein, crystal structure, filovirus, assembly mechanism

Section: Discussionmentioning

confidence: 99%

Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 Peptide in Marburg Virus

Liu

Dong

et al. 2017

“…HAZV and CCHFV exhibit the same pathology in virus-inoculated immune-deficient animals (8,9) and share susceptibility to ribavirin (7,10,11). The N protein of HAZV structurally resembles that of CCHFV (12), and both proteins interact with heat shock protein 70 family members for efficient viral growth (13). HAZV, then, may be a suitable model to examine the molecular basis of pathogenic-nairovirus replication.…”

mentioning

confidence: 99%

A Minigenome Study of Hazara Nairovirus Genomic Promoters

Matsumoto

Ohta

Kolakofsky

et al. 2019

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales. The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis. IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3′ and 5′ strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

“…1A). Previous work by us and others (16,19) established that the generation of 30-and 22-kDa products resulted from caspase-3 cleavage of HAZV-N at a consensus DQVD motif (Fig. 1B) located at the apex of the HAZV-N arm domain (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…Rescue of infectious HAZV variants with alterations to caspase cleavage sites on the N protein arm apex. Previous work by us and others identified caspase-3 cleavage motifs on both the CCHFV-N (DEVD) and HAZV-N (DQVD) proteins, at the apex of their respective arm domains (16,17,19), and mapping of these sites onto the corresponding crystal structures showed these are in precisely superimposable positions ( Fig. 5A).…”

Section: Confirmation Of Hazv Rescue From a Cdna Source By Incorporatmentioning

confidence: 83%

“…We and others recently solved the crystal structure of the N proteins of both HAZV and CCHFV, which revealed the location of a conserved caspase-3 cleavage (C3C) site on the exposed apex of the extended arm domain, suggestive of an important role at some stage of the nairovirus multiplication cycle (16)(17)(18)(19). One possibility was that the C3C site acted as a caspase-3 decoy, diverting caspase-3 away from its native cellular substrates and thus preventing or prolonging apoptosis activation, as recently found for the related Junin arenavirus (JUNV) (20).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage

Fuller

Surtees

Slack

et al. 2019

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical role aside from caspase cleavage. IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.