Hazara Nairovirus Requires COPI Components in both Arf1-Dependent and Arf1-Independent Stages of Its Replication Cycle.

Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex.

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

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.

Hazara nairovirus elicits differential induction of apoptosis and nucleocapsid protein cleavage in mammalian and tick cells.

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.

Expression, purification and crystallization of the Crimean-Congo haemorrhagic fever virus nucleocapsid protein.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus within the Bunyaviridae family of segmented negative-sense RNA viruses. This paper describes the expression, purification and crystallization of full-length CCHFV nucleocapsid (N) protein and the collection of a 2.1 Šresolution X-ray diffraction data set using synchrotron radiation. Crystals of the CCHFV N protein belonged to space group C2, with unit-cell parameters a = 150.38, b = 72.06, c = 101.23 Å, ß = 110.70° and two molecules in the asymmetric unit. Circular-dichroism analysis provided insight into the secondary structure, whilst gel-filtration analysis revealed possible oligomeric states of the N protein. Structural determination is ongoing.

The VSV polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation.

Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bi-cistronic replicons were constructed containing an mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed.

Transcription and replication of nonsegmented negative-strand RNA viruses.

The nonsegmented negative-strand (NNS) RNA viruses of the order Mononegavirales include a wide variety of human, animal, and plant pathogens. The NNS RNA genomes of these viruses are templates for two distinct RNA synthetic processes: transcription to generate mRNAs and replication of the genome via production of a positive-sense antigenome that acts as template to generate progeny negative-strand genomes. The four virus families within the Mononegavirales all express the information encoded in their genomes by transcription of discrete subgenomic mRNAs. The key feature of transcriptional control in the NNS RNA viruses is entry of the virus-encoded RNA-dependent RNA polymerase at a single 3' proximal site followed by obligatory sequential transcription of the linear array of genes. Levels of gene expression are primarily regulated by position of each gene relative to the single promoter and also by cis-acting sequences located at the beginning and end of each gene and at the intergenic junctions. Obligatory sequential transcription dictates that termination of each upstream gene is required for initiation of downstream genes. Therefore, termination is a means to regulate expression of individual genes within the framework of a single transcriptional promoter. By engineering either whole virus genomes or subgenomic replicon derivatives, elements important for signaling transcript initiation, 5' end modification, 3' end polyadenylation, and transcription termination have been identified. Although the diverse families of NNS RNA virus use different sequences to control these processes, transcriptional termination is a common theme in controlling gene expression and overall transcriptional regulation is key in controlling the outcome of viral infection. The latest models for control of replication and transcription are discussed.

Polymerase slippage at vesicular stomatitis virus gene junctions to generate poly(A) is regulated by the upstream 3'-AUAC-5' tetranucleotide: implications for the mechanism of transcription termination.

Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3'-AUAC-5', the U(7) tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3' poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U(7) tract. Previously we determined that in addition to the AUAC tetranucleotide, the U(7) tract was an essential signal in the termination process. Shortening or interrupting the U(7) tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U(7) tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U(7) tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U(7) tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis.

Identification of a minimal size requirement for termination of vesicular stomatitis virus mRNA: implications for the mechanism of transcription.

The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3'-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5' modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.

Role of the intergenic dinucleotide in vesicular stomatitis virus RNA transcription.

To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in modulation of polymerase activity, we analyzed the RNA synthesis activities of bicistrionic genomic analogs that contained either the authentic N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide intergenic sequences, or no intergenic sequence at all. Quantitative measurements of the amounts of upstream, downstream, and readthrough mRNAs that were transcribed by these mutant templates showed that the behavior of the viral polymerase was profoundly affected by the nucleotide sequence that it encountered as it traversed the gene junction, although the polymerase was able to accommodate a remarkable degree of sequence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic sequence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation of downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analogs with altered intergenic sequences directed abundant synthesis of a readthrough transcript without correspondingly high levels of downstream mRNA, an observation inconsistent with the shunting model of VSV transcription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findings of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.