SARS-CoV-2-Virus structure and life cycle.

This book chapter presents a concise overview of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. It explores viral classification based on morphology and nucleic acid composition with a focus on DNA and RNA viruses, the SARS-CoV-2 structure including the structural as well as nonstructural proteins in detail, and the viral replication mechanisms. The chapter then delves into the characteristics and diversity of coronaviruses, particularly SARS-CoV-2, highlighting its similarities with other beta-coronaviruses. The replication and transcription complex, RNA elongation, and capping, as well as the role of accessory proteins in viral replication and modulation of the host immune response is discussed extensively.

Missense mutations in spike protein of SARS-CoV-2 delta variant contribute to the alteration in viral structure and interaction with hACE2 receptor.

INTRODUCTION: Many of the global pandemics threaten human existence over the decades among which coronavirus disease (COVID-19) is the newest exposure circulating worldwide. The RNA encoded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is referred as the pivotal agent of this deadly disease that induces respiratory tract infection by interacting host ACE2 receptor with its spike glycoprotein. Rapidly evolving nature of this virus modified into new variants helps in perpetrating immune escape and protection against host defense mechanism. Consequently, a new isolate, delta variant originated from India is spreading perilously at a higher infection rate. METHODS: In this study, we focused to understand the conformational and functional significance of the missense mutations found in the spike glycoprotein of SARS-CoV-2 delta variant performing different computational analysis. RESULTS: From physiochemical analysis, we found that the acidic isoelectric point of the virus elevated to basic pH level due to the mutations. The targeted mutations were also found to change the interactive bonding pattern and conformational stability analyzed by the molecular dynamic's simulation. The molecular docking study also revealed that L452R and T478K mutations found in the RBD domain of delta variant spike protein contributed to alter interaction with the host ACE2 receptor. CONCLUSIONS: Overall, this study provided insightful evidence to understand the morphological and attributive impact of the mutations on SARS-CoV-2 delta variant.

Superimposition of Viral Protein Structures: A Means to Decipher the Phylogenies of Viruses.

Superimposition of protein structures is key in unravelling structural homology across proteins whose sequence similarity is lost. Structural comparison provides insights into protein function and evolution. Here, we review some of the original findings and thoughts that have led to the current established structure-based phylogeny of viruses: starting from the original observation that the major capsid proteins of plant and animal viruses possess similar folds, to the idea that each virus has an innate "self". This latter idea fueled the conceptualization of the PRD1-adenovirus lineage whose members possess a major capsid protein (innate "self") with a double jelly roll fold. Based on this approach, long-range viral evolutionary relationships can be detected allowing the virosphere to be classified in four structure-based lineages. However, this process is not without its challenges or limitations. As an example of these hurdles, we finally touch on the difficulty of establishing structural "self" traits for enveloped viruses showcasing the coronaviruses but also the power of structure-based analysis in the understanding of emerging viruses.

Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication.

Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required.IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.

Detection of viral components in exosomes derived from NDV-infected DF-1 cells and their promoting ability in virus replication.

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection.

Bluetongue virus structure and assembly.

Bluetongue virus (BTV) is an insect-vectored emerging pathogen of wild ruminants and livestock in many parts of the world. The virion particle is a complex structure of consecutive layers of protein surrounding a genome of ten double-stranded (ds) RNA segments. BTV has been studied as a model system for large, non-enveloped dsRNA viruses. Several new techniques have been applied to define the virus-encoded enzymes required for RNA replication to provide an order for the assembly of the capsid shell and the protein sequestration required for it. Further, a reconstituted in vitro system has defined the individual steps of the assembly and packaging of the genomic RNA. These findings illuminate BTV assembly and indicate the pathways that related viruses might use to provide an informed starting point for intervention or prevention.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment.

Tail morphology controls DNA release in two Salmonella phages with one lipopolysaccharide receptor recognition system.

Bacteriophages use specific tail proteins to recognize host cells. It is still not understood to molecular detail how the signal is transmitted over the tail to initiate infection. We have analysed in vitro DNA ejection in long-tailed siphovirus 9NA and short-tailed podovirus P22 upon incubation with Salmonella typhimurium lipopolysaccharide (LPS). We showed for the first time that LPS alone was sufficient to elicit DNA release from a siphovirus in vitro. Crystal structure analysis revealed that both phages use similar tailspike proteins for LPS recognition. Tailspike proteins hydrolyse LPS O antigen to position the phage on the cell surface. Thus we were able to compare in vitro DNA ejection processes from two phages with different morphologies with the same receptor under identical experimental conditions. Siphovirus 9NA ejected its DNA about 30 times faster than podovirus P22. DNA ejection is under control of the conformational opening of the particle and has a similar activation barrier in 9NA and P22. Our data suggest that tail morphology influences the efficiencies of particle opening given an identical initial receptor interaction event.

Critical role of leucine-valine change in distinct low pH requirements for membrane fusion between two related retrovirus envelopes.

Many viruses use a pH-dependent pathway for fusion with host cell membrane, the mechanism of which is still poorly understood. Here we report that a subtle leucine (Leu)-valine (Val) change at position 501 in the envelope glycoproteins (Envs) of two related retroviruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), is responsible for their distinct low pH requirements for membrane fusion and infection. The Leu and Val residues are predicted to reside within the C-terminal heptad repeat (HR2) region of JSRV and ENTV Envs, particularly proximal to the hairpin turn of the putative six-helix bundle (6HB). Substitution of the JSRV Leu with a Val blocked the Env-mediated membrane fusion at pH 5.0, whereas replacement of the ENTV Val with a Leu rendered the ENTV Env capable of fusing at pH 5.0. A Leu-Val change has no apparent effect on the stability of native Env, but appears to stabilize an intermediate induced by receptor binding. These results are consistent with the existence of at least two metastable conformations of these viral glycoproteins, the native prefusion conformation and a receptor-induced metastable intermediate. Collectively, this work represents an interesting perhaps unique example whereby a simple Leu-Val change has critical impact on pH-dependent virus fusion and entry.