We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.

The goal of a decision-based adversarial attack on a trained model is to generate adversarial examples based solely on observing output labels returned by the targeted model. We develop HopSkipJumpAttack, a family of algorithms based on a novel estimate of the gradient direction using binary information at the decision boundary. The proposed family includes both untargeted and targeted attacks optimized for 2 and ∞ similarity metrics respectively. Theoretical analysis is provided for the proposed algorithms and the gradient direction estimate. Experiments show HopSkipJumpAttack requires significantly fewer model queries than several state-of-the-art decision-based adversarial attacks. It also achieves competitive performance in attacking several widely-used defense mechanisms.

The dynamics and regulation of HIV-1 nuclear import and its intranuclear movements after import have not been studied. To elucidate these essential HIV-1 post-entry events, we labeled viral complexes with two fluorescently tagged virion-incorporated proteins (APOBEC3F or integrase), and analyzed the HIV-1 dynamics of nuclear envelope (NE) docking, nuclear import, and intranuclear movements in living cells. We observed that HIV-1 complexes exhibit unusually long NE residence times (1.5±1.6 hrs) compared to most cellular cargos, which are imported into the nuclei within milliseconds. Furthermore, nuclear import requires HIV-1 capsid (CA) and nuclear pore protein Nup358, and results in significant loss of CA, indicating that one of the viral core uncoating steps occurs during nuclear import. Our results showed that the CA-Cyclophilin A interaction regulates the dynamics of nuclear import by delaying the time of NE docking as well as transport through the nuclear pore, but blocking reverse transcription has no effect on the kinetics of nuclear import. We also visualized the translocation of viral complexes docked at the NE into the nucleus and analyzed their nuclear movements and determined that viral complexes exhibited a brief fast phase (<9 min), followed by a long slow phase lasting several hours. A comparison of the movement of viral complexes to those of proviral transcription sites supports the hypothesis that HIV-1 complexes quickly tether to chromatin at or near their sites of integration in both wild-type cells and cells in which LEDGF/p75 was deleted using CRISPR/cas9, indicating that the tethering interactions do not require LEDGF/p75. These studies provide novel insights into the dynamics of viral complex-NE association, regulation of nuclear import, viral core uncoating, and intranuclear movements that precede integration site selection.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.RNA replication by positive-strand RNA viruses is closely associated with cellular membranes. For all well-studied eukaryotic positive-strand RNA viruses, the viral RNA-dependent RNA replication complex copurifies with membrane extracts from infected cells (8,9,14,18,43). In vivo and in vitro studies with positive-strand RNA viruses suggest that membrane association is essential for at least some steps of RNA replication (7,38,58). In some cases, negative-strand RNA synthesis activity can be solubilized from membranes (24,43,57,58). However, in vivo, both positive-and negative-strand RNA synthesis occurs in membrane-associated complexes (10,45,46). The membrane interactions of replication factors from most viruses appear specific in that the replication complexes of different positive-strand RNA viruses associate with different intracellular membranes (18,19,41,51,52). However, the mechanisms by which such viral replication complexes are targeted to and assembled on specific membrane sites remain poorly understood.Brome mosaic virus (BMV), the type member of the Bromovirus genus, is a positive-strand RNA virus in the alphavirus-like superfamily (1). The BMV genome is composed of three RNAs. RNA3 encod...