Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching Endonucleases

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase

Structures of influenza A virus RNA polymerase offer insight into viral genome replication

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3,4,5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication—which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7,8,9,10—remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0–4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3′ cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs

Understanding Influenza

Influenza, a serious illness of humans and domesticated animals, has been studied intensively for many years. It therefore provides an example of how much we can learn from detailed studies of an infectious disease and of how even the most intensive scientific research leaves further questions to answer. This introduction is written for researchers who have become interested in one of these unanswered questions, but who may not have previously worked on influenza. To investigate these questions, researchers must not only have a firm grasp of relevant methods and protocols; they must also be familiar with the basic details of our current understanding of influenza. This article therefore briefly covers the burden of disease that has driven influenza research, summarizes how our thinking about influenza has evolved over time, and sets out key features of influenza viruses by discussing how we classify them and what we understand of their replication. It does not aim to be comprehensive, as any researcher will read deeply into the specific areas that have grasped their interest. Instead, it aims to provide a general summary of how we came to think about influenza in the way we do now, in the hope that the reader’s own research will help us to understand it better

Firefly genomes illuminate parallel origins of bioluminescence in beetles

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle

Structure of the RNA-dependent RNA polymerase from influenza C virus

The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.</p

Structure of the RNA-dependent RNA polymerase from influenza C virus

The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 &Aring;. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 &Aring; away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180&deg; from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.This thesis is not currently available in ORA

RNA-free and ribonucleoprotein-associated influenza virus polymerases directly bind the serine-5 phosphorylated carboxyl-terminal domain of host RNA polymerase II

Influenza viruses subvert the transcriptional machinery of their hosts to synthesise their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap-snatching, the virus steals short 5ʹ capped RNA fragments from host capped RNAs and uses these to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5 phosphorylated form of the Pol II CTD, both in the presence and absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap-snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation

The surface-exposed PA51-72-loop of the influenza A virus polymerase is required for viral genome replication

The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2 and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro. Furthermore, vRNA synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilisation of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular

The surface-exposed PA51-72-loop of the influenza A virus polymerase is required for viral genome replication

The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2 and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro. Furthermore, vRNA synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilisation of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular