Evaluating RNA Structural Flexibility: Viruses Lead the Way.

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis.

Being opportunistic intracellular pathogens, viruses are dependent on the host for their replication. They hijack host cellular machinery for their replication and survival by targeting crucial cellular physiological pathways, including transcription, translation, immune pathways, and apoptosis. Immediately after translation, the host and viral proteins undergo a process called post-translational modification (PTM). PTMs of proteins involves the attachment of small proteins, carbohydrates/lipids, or chemical groups to the proteins and are crucial for the proteins' functioning. During viral infection, host proteins utilize PTMs to control the virus replication, using strategies like activating immune response pathways, inhibiting viral protein synthesis, and ultimately eliminating the virus from the host. PTM of viral proteins increases solubility, enhances antigenicity and virulence properties. However, RNA viruses are devoid of enzymes capable of introducing PTMs to their proteins. Hence, they utilize the host PTM machinery to promote their survival. Proteins from viruses belonging to the family: Togaviridae, Flaviviridae, Retroviridae, and Coronaviridae such as chikungunya, dengue, zika, HIV, and coronavirus are a few that are well-known to be modified. This review discusses various host and virus-mediated PTMs that play a role in the outcome during the infection.

Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins.

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

Novel modulators of p53-signaling encoded by unknown genes of emerging viruses.

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs.

Structure and Expression of Large (+)RNA Genomes of Viruses of Higher Eukaryotes.

Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses - animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5'-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses (i.e., coronaviruses) contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3'-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size.

Coronavirus disease 2019 (COVID-19), human erythrocytes and the PKC-alpha/-beta inhibitor chelerythrine -possible therapeutic implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. Until now, diverse drugs have been used for the treatment of COVID-19. These drugs are associated with severe side effects, e.g. induction of erythrocyte death, named eryptosis. This massively affects the oxygen (O2) supply of the organism. Therefore, three elementary aspects should be considered simultaneously: (1) a potential drug should directly attack the virus, (2) eliminate virus-infected host cells and (3) preserve erythrocyte survival and functionality. It is known that PKC-α inhibition enhances the vitality of human erythrocytes, while it dose-dependently activates the apoptosis machinery in nucleated cells. Thus, the use of chelerythrine as a specific PKC-alpha and -beta (PKC-α/-ß) inhibitor should be a promising approach to treat people infected with SARS-CoV-2.

TAM receptors: A phosphatidylserine receptor family and its implications in viral infections.

Phosphatidylserine (PS) is an anionic phospholipid that is usually localized in the inner leaflets of the plasma membrane. However, the enzyme scramblase catalyzes the externalization of PS on the outer leaflet of the plasma membrane during apoptosis or cellular stress. This event prompts the recognition of PS displaying cells by phagocytes leading to "apoptotic clearance." Multiple PS receptors (PSRs) mediate this process including members from the TAM (Tyro3, Axl, Mertk) receptor Tyrosine kinases (RTKs) by interacting with PS via bridging proteins like Gas6 and ProS1. Ironically, this network (PS/TAM) that evolved for boosting cellular health through clearance of apoptotic and necrotic cells, has been manoeuvred by pathogens and tumor cells using "apoptotic mimicry." Enveloped viruses, responsible for most of the lethal epidemics and pandemics including the current SARS-CoV2 outbreak, have employed apoptotic mimicry to their advantage. In the current chapter, we summarize the existing knowledge regarding the involvement of PS/Gas6, ProS1/TAM in facilitating infectivity in a diverse set of cell lines, animals as well as organoids. This network executes a largely proviral role in facilitating infection as seen with Zika, Ebola, Influenza and Dengue viruses. However, this response varies with strains and the cells infected, and in some cases, this same signaling displays an antiviral function. We also report multiple studies that have used neutralizing antibodies and small molecule inhibitors in successfully reducing viral replication and ameliorating pathogenicity. Knowledge about this unique signaling pathway and measures that can be taken to inhibit it is most valuable now given how enveloped viruses lead to plagues on the entire globe.

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

SUMO and Cytoplasmic RNA Viruses: From Enemies to Best Friends.

SUMO is a ubiquitin-like protein that covalently binds to lysine residues of target proteins and regulates many biological processes such as protein subcellular localization or stability, transcription, DNA repair, innate immunity, or antiviral defense. SUMO has a critical role in the signaling pathway governing type I interferon (IFN) production, and among the SUMOylation substrates are many IFN-induced proteins. The overall effect of IFN is increasing global SUMOylation, pointing to SUMO as part of the antiviral stress response. Viral agents have developed different mechanisms to counteract the antiviral activities exerted by SUMO, and some viruses have evolved to exploit the host SUMOylation machinery to modify their own proteins. The exploitation of SUMO has been mainly linked to nuclear replicating viruses due to the predominant nuclear localization of SUMO proteins and enzymes involved in SUMOylation. However, SUMOylation of numerous viral proteins encoded by RNA viruses replicating at the cytoplasm has been lately described. Whether nuclear localization of these viral proteins is required for their SUMOylation is unclear. Here, we summarize the studies on exploitation of SUMOylation by cytoplasmic RNA viruses and discuss about the requirement for nuclear localization of their proteins.