Structural insight into arenavirus replication machinery.

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.

RNA ligands activate the Machupo virus polymerase and guide promoter usage.

Segmented negative-sense (SNS) RNA viruses initiate infection by delivering into cells a suite of genomic RNA segments, each sheathed by the viral nucleocapsid protein and bound by the RNA-dependent RNA-polymerase (RdRP). For the orthomyxovirus influenza and the bunyavirus La Crosse, the 5' end of the genomic RNA binds as a hook-like structure proximal to the active site of the RdRP. Using an in vitro assay for the RNA-dependent RNA-polymerase (RdRP) of the arenavirus Machupo (MACV), we demonstrate that the 5' genomic and antigenomic RNAs of both small and large genome segments stimulate activity in a promoter-specific manner. Functional probing of the activating RNAs identifies intramolecular base-pairing between positions +1 and +7 and a pseudotemplated 5' terminal guanine residue as key for activation. Binding of structured 5' RNAs is a conserved feature of all SNS RNA virus polymerases, implying that promoter-specific RdRP activation extends beyond the arenaviruses. The 5' RNAs and the RNA binding pocket itself represent targets for therapeutic intervention.

Structures of arenaviral nucleoproteins with triphosphate dsRNA reveal a unique mechanism of immune suppression.

A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3'-5' exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

Assembly of a functional Machupo virus polymerase complex.

Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5' cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2-5 in the 3' terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex assembly to occur. Our results provide a model for arenavirus polymerase-template interactions and reveal the structural organization of a negative-strand RNA virus L protein.

Distinctive RNA transcriptase, polyadenylic acid polymerase, and polyuridylic acid polymerase activities associated with Pichinde virus.

Three RNA polymerase activities were found and associated with purified Pichinde virus, a member of the Arenaviridae. A heat-labile polymerase activity which required all four ribonucleoside triphosphates for optimal activity co-sedimented on sucrose gradient centrifugation with the viral ribonucleoprotein complex from detergent-disrupted virus preparations. This enzyme synthesized heteropolymers which represented about 23% of the genome RNA as determined by nucleic acid hybridization. Two relatively heat-stable polymerase activities which differed in their cation requirement and substrate specificity were recovered with the virus-associated ribosomes. These polymerase activities synthesized homopolymers of limited chain length: in the presence of 10 mM Mg2%, polyuridylic acid was made, whereas in the presence of 1 mM Mn2%, polyadenylic acid was made. The addition of complementary RNA synthesized with the viral transcriptase in vitro to the reaction mixture containing the polyadenylic acid polymerase activity resulted in the terminal addition of polyadenylic acid to the complementary RNA. The possible function of the ribosome-associated polymerase activities in the replication of the virus is discussed.