The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20^L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 20⁴ = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve.

Nasopharyngeal carcinoma, Kaposi's sarcoma, and B-cell lymphomas are human malignancies associated with gammaherpesvirus infections. Members of this virus family are characterized by their ability to establish latent infections in lymphocytes. The latent viral genome expresses very few gene products. The infected cells are therefore poorly recognized by the host immune system, allowing the virus to persist for long periods of time. We sought to identify the cell-specific factors that allow these viruses to redirect their life cycle from productive replication to latency. We find that the cellular transcription factor NF-κB can regulate this process. Epithelial cells and fibroblasts support active (lytic) gammaherpesvirus replication and have low NF-κB activity. However, overexpression of NF-κB in these cells inhibits the replication of the gammaherpesvirus murine herpesvirus 68 (MHV68). In addition, overexpression of NF-κB inhibits the activation of lytic promoters from MHV68 and human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). In lymphocytes latently infected with KSHV or EBV, the level of NF-κB activity is high, and treatment of these cells with an NF-κB inhibitor leads to lytic protein synthesis consistent with virus reactivation. These results suggest that high levels of NF-κB can inhibit gammaherpesvirus lytic replication and may therefore contribute to the establishment and maintenance of viral latency in lymphocytes. They also suggest that NF-κB may be a novel target for the disruption of virus latency and therefore the treatment of gammaherpesvirus-related malignancies.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.

A high-resolution structure of the bacteriophage MS2 sheds light on the structure of the genome and how the genome is delivered into a bacterium. Hong Zhou and colleagues provide the first description of genome–capsid interactions in a spherical single-stranded RNA (ssRNA) virus, using the bacteriophage MS2 as a model. Unlike double-stranded DNA viruses that pump their genome into a preformed capsid, ssRNA viruses co-assemble their capsid with their genome. Here the authors determine the MS2 structure at 3.6 Å resolution and are able to trace around 80% of the backbone of the viral genome, identifying regions that react with the maturation protein and providing insights into the ssRNA capsid co-assembly process. Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid1,2,3, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome4,5,6,7; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus)8; it was the first fully sequenced organism9 and is a model system for studies of translational gene regulation10,11, RNA–protein interactions12,13,14, and RNA virus assembly15,16,17. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice18,19. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.