Ana S. Martins

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2′-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA–RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

West Nile and dengue viruses are closely related flaviviruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their capsid proteins sequence and structure are particularly similar, forming highly superimposable α-helical homodimers. Measuring protein-ligand interactions at the single-molecule level yields detailed information of biological and biomedical relevance. In this work, such an approach was successfully applied on the characterization of the West Nile virus capsid protein interaction with host lipid systems, namely intracellular lipid droplets (an essential step for dengue virus replication) and blood plasma lipoproteins. Dynamic light scattering measurements show that West Nile virus capsid protein binds very low-density lipoproteins, but not low-density lipoproteins, and this interaction is dependent of potassium ions. Zeta potential experiments show that the interaction with lipid droplets is also dependent of potassium ions as well as surface proteins. The forces involved on the binding of the capsid protein with lipid droplets and lipoproteins were determined using atomic force microscopy-based force spectroscopy, proving that these interactions are K⁺-dependent rather than a general dependence of ionic strength. The capsid protein interaction with host lipid systems may be targeted in future therapeutic strategies against different flaviviruses. The biophysical and nanotechnology approaches employed in this study may be applied to characterize the interactions of other important proteins from different viruses, in order to understand their life cycles, as well as to find new strategies to inhibit them.

Lipid droplets (LDs) are intracellular organelles for neutral lipid storage, originated from the endoplasmic reticulum (ER). These organelles are composed by a neutral core of triacylglycerols and sterol esters, surrounded by a monolayer of phospholipids and cholesterol, with several proteins at the surface. LDs play a central role in cellular energy storage and lipid metabolism. Viruses from the Flaviviridae family, namely dengue virus (DENV), hepatitis C virus (HCV), West Nile virus and Zika virus, interact with LDs to usurp the host lipid metabolism for their own viral replication and pathogenesis. Flaviviruses have a similar structure, with a lipid bilayer, where the envelope and membrane proteins are located, surrounding the nucleocapsid. The nucleocapsid contains the RNA genome complexed with multiple copies of the capsid protein. During viral replication, these positive-sense RNA viruses hijack the infected cell intracellular membrane machinery, increasing the number of host intracellular LDs and their diameter. LDs are targeted by several viral proteins and play multiple roles during the viral life cycle. The core and capsid protein, of HCV and DENV, respectively, extensively interact with LDs to regulate essential steps for their replication and assembly. In fact, flaviviruses use their viral proteins to regulate viral RNA replication through the interaction with LDs-surface proteins and use LDs, near the endoplasmic reticulum, as platforms for virus assembly. A lack of knowledge of key viral life cycle steps, such as assembly and encapsidation, is one of the reasons for the absence of effective vaccines or specific therapeutics against important pathogens from the Flaviviridae family. Detailed studies of LDs and their interactions with viral factors are crucial for the development of possible inhibitors of these key steps of viral replication. Microscopy studies have been the approach commonly used to observe LDs accumulation and localization in infected cell cultures. However, there are other approaches that enable the study of these organelles in terms of size, surface charge and molecular interactions. Here, we review different biophysical techniques that can be used to extract this information, as well as approaches to isolate and purify LDs from a cell culture.