The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication

An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5′-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5′ guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5′ terminus of the viral genome to initiate RNA synthesis at the 3′ terminus.

Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

Multiple Capsid Protein Binding Sites Mediate Selective Packaging of the Alphavirus Genomic RNA

ABSTRACTThe alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, we comprehensively defined binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrated Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments showed that PS was not required for production of infectious SFV or Chikungunya virus. Instead, we identified multiple novel Cp binding sites that were enriched on gRNA-specific regions and promoted infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrated that budding caused discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site was maintained throughout virus assembly, and specifically bound and assembled with Cp into core-like particles in vitro. Together our data suggest a new model for selective alphavirus genome recognition and assembly.

Single-step label-free hepatitis B virus detection by a piezoelectric biosensor

Probe densityvs.genome recognition selectivity.

DDB2 promotes chromatin decondensation at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.