Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ₂-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ₂-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence. Kaposi sarcoma-associated herpesvirus (KSHV) causes Kaposi Sarcoma, Primary Effusion lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Its oncogenic effect is linked to its ability to persist in a latent form for the life time of infected individuals. During latency viral genomes are replicated and passed to daughter cells in synchrony with the infected cell without the formation of new virions. A key viral protein in this process is the latency-associated nuclear antigen, LANA. In latently infected cells, viral genomes and LANA form characteristic nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin components. We have solved the crystal structure of the c-terminal, DNA-binding, domain (CTD) of KSHV LANA (kLANA) and its homologue mLANA of a related murine γ₂-herpesvirus, which is frequently used as a model to study latent persistence in vivo. We also identified the binding site for two chromatin proteins, BRD2/4, by NMR spectroscopy. We demonstrate the functional importance of these structural features, and their contribution to latent replication and ‘LANA speckle’ formation, in cell culture and in vivo experiments. Our results provide a structural basis for the assembly of LANA-containing nuclear structures that are required for latent viral replication and persistence.

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.

Significance: KSHV is the etiological agent of Kaposi sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. During latency, this dsDNA tumor virus expresses only a small subset of its more than 90 ORFs. Among these is ORF73/latency-associated nuclear antigen (LANA), which acts as the origin binding protein and chromatin anchor of the extrachromosomal viral genome. This work provides detailed structural insights into the DNA-binding characteristics of LANA. We also report a previously unrecognized, third LANA binding site within the minimal replicator of Kaposi sarcoma herpesvirus. In addition to its mechanistic implications for latent viral persistence, the X-ray crystal structure of LANA bound to LANA binding site 1 DNA may assist in the tailored development of therapeutic LANA inhibitors.

A block to viral cell entry: Crimean-Congo hemorrhagic fever virus is a tickborne virus that can cause severe disease and even death in humans. Disease occurrence is linked to the geographic range of the tick vector, and climate change may increase this range. Infection of host cells requires the fusion glycoprotein Gc, which is the main target of neutralizing antibodies. Mishra et al . build on previous work that identified a combination of two Gc-targeting antibodies that gave postexposure protection in an animal model. The authors determined the structure of the antigen-binding fragments of the two antibodies bound to a prefusion form of Gc and also the structure of Gc after the conformational change into the trimeric postfusion form. The structures show how the antibodies work together to block membrane fusion. —VV

Rabies is a severe viral infection that causes an acute encephalomyelitis, which presents a case fatality of nearly 100% after the manifestation of neurological clinical signs. Rabies can be efficiently prevented with post-exposure prophylaxis (PEP), composed of vaccines and anti-rabies immunoglobulins (RIGs); however, no treatment exists for symptomatic rabies. The PEP protocol faces access and implementation obstacles in resource-limited settings, which could be partially overcome by substituting RIGs for monoclonal antibodies (mAbs). mAbs offer lower production costs, consistent supply availability, long-term storage/stability, and an improved safety profile. Here we summarize the key features of the different available mAbs against rabies, focusing on their application in PEP and highlighting their potential in a novel therapeutic approach.

Rabies virus (RABV) causes fatal encephalitis in more than 59,000 people yearly. Upon the bite of an infected animal, the development of clinical disease can be prevented with post-exposure prophylaxis (PEP), which includes the administration of Rabies immunoglobulin (RIG). However, the high cost and limited availability of serum-derived RIG severely hamper its wide use in resource-limited countries. A safe low-cost alternative is provided by using broadly neutralizing monoclonal antibodies (bnAbs). Here we report the X-ray structure of one of the most potent and most broadly reactive human bnAbs, RVC20, in complex with its target domain III of the RABV glycoprotein (G). The structure reveals that the RVC20 binding determinants reside in a highly conserved surface of G, rationalizing its broad reactivity. We further show that RVC20 blocks the acid-induced conformational change required for membrane fusion. Our results may guide the future development of direct antiviral small molecules for Rabies treatment.