Isolation of reconstructed functional ribonucleoprotein complexes of Machupo virus.

ABSTRACT: Arenaviruses initiate infection by delivering a transcriptionally-competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (vRNA) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA, affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodisperse L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase - which copies the viral genome segments and synthesizes all four viral mRNAs - bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery, and thus advancing antiviral strategies targeting this essential complex.

SUBMITTER: Pyle JD 

PROVIDER: S-EPMC8549503 | biostudies-literature |

REPOSITORIES: biostudies-literature