SARS-CoV-2 Proteins: Are They Useful as Targets for COVID-19 Drugs and Vaccines?

The proteins of coronavirus are classified as non-structural, structural, and accessory. There are 16 non-structural viral proteins besides their precursors (1a and 1ab polyproteins). The non-structural proteins are named nsp1 to nsp16, and they act as enzymes, coenzymes, and binding proteins to facilitate the replication, transcription, and translation of the virus. The structural proteins are bound to the RNA in the nucleocapsid (N- protein) or to the lipid bilayer membrane of the viral envelope. The lipid bilayer proteins include the membrane protein (M), an envelope protein (E), and spike protein (S). Besides their role as structural proteins, they are essential for the host cells' binding and invasion. The SARS-CoV-2 contains six accessory proteins which participate in the viral replication, assembly and virus-host interactions. The SARS-CoV-2 accessory proteins are orf3a, orf6, orf7a, orf7b, orf8, and orf10. The functions of the SARS-CoV-2 are not well known, while the functions of their corresponding proteins in SARS-CoV are either well known or poorly studied. Recently, the Oxford University and Astrazeneca, Pfizer and BioNTech have made SARS-CoV-2 vaccines by targeting the spike protein gene. The US Food and Drug Administration (FDA) and the health authorities of the United Kingdom have approved and started conducting vaccinations using the Pfizer and BioNTech mRNA vaccine. Also, The FDA of the USA has approved the use of two monoclonal antibodies produced by Regeneron pharmaceuticals to target the spike protein for treating COVID-19. The SARS-CoV-2 proteins can be used for the diagnosis, as drug targets and in vaccination trials for COVID-19. In future COVID-19 research, more efforts should be made to elaborate the functions and structure of the SARS-CoV- 2 proteins so as to use them as targets for COVID-19 drugs and vaccines. Special attention should be paid to extensive research on the SARS-CoV-2 nsp3, orf8, and orf10.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.

Introduction of direct-acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral-targeting compounds, is limited because HBV requires only four viral genes for its efficient replication/infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell-based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, whereas it did not affect the NTCP-mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan-genotypic anti-HBV activity and was also effective against a clinically relevant nucleoside analog-resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by cotreatment. Moreover, derivative analysis could identify small molecules that demonstrated more-potent anti-HBV activity over PAC. CONCLUSION: PAC and its analogs represent a new class of anti-HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well-tolerated, and broadly active inhibitor of HBV infection. (Hepatology 2017;65:1104-1116).

DNA shuffling of the GP3 genes of porcine reproductive and respiratory syndrome virus (PRRSV) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen. Here we applied the DNA shuffling approaches to molecularly breed the PRRSV GP3 gene, a neutralizing antibodies inducer, in an attempt to improve its heterologous cross-neutralizing ability. The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling. Additionally, synthetic DNA shuffling of the GP3 gene was also performed using degenerate oligonucleotides. The shuffled-GP3-libraries were cloned into the backbone of a DNA-launched PRRSV infectious clone pIR-VR2385-CA. Four traditional-shuffled chimeras each representing all 6 parental strains and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication both in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain FL-12.

Small peptide inhibitors disrupt a high-affinity interaction between cytoplasmic dynein and a viral cargo protein.

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.

Defective rotavirus particle assembly in lovastatin-treated MA104 cells.

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in "empty-looking" rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion.

Interaction and assembly of HBV structural proteins: novel target sites of anti-HBV agents.

Human hepatitis B virus (HBV) causes chronic hepatitis disease which is a major public health problem worldwide. HBV has 4 genes encoding viral DNA polymerase, protein X and two structural proteins, the surface and core proteins. HBV DNA polymerase has been a primary target for the development of anti-HBV agents due to its enzymatic nature, and several nucleoside derivatives that inhibit HBV polymerase are currently used as anti-HBV therapeutics. On the other hand, accumulating information on the capsid assembly and the maturation process of HBV particles provides additional approaches for the development of anti-HBV agents. Proper interaction between core proteins is required for assembly of the nucleocapsid, and the specificity of the interactions between the capsid and surface proteins is essential for the maturation of active HBV in infected cells. In this article, the assembly process of active HBV particles and approaches to utilize the interactions of HBV structural proteins as target site for the development of anti-HBV agents are reviewed. In particular, novel approaches to target the assembly process and the interaction between HBV structural proteins are introduced.

Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i). an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii). a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii). an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Inhibition of type 5 adenovirus infectivity by periodate oxidation.

Periodate oxidation of purified type 5 Adenovirus (Ad5) led to a mean loss of infectivity of 6.84 logs. There were no significant differences in adsorption and penetration between oxidized and mock-oxidized virus. However, after infection with oxidized virus, no synthesis of viral structural proteins could be detected and a 78.5% inhibition of viral DNA synthesis was observed. Labelling experiments performed by treating oxidized and mock-oxidized virus with tritiated sodium borohydride revealed that the fiber glycoprotein was one of the proteins labelled in oxidized virus whereas no labelled proteins were detected in non oxidized virus. In addition, it was found that one mol of formaldehyde generated during oxidation of sugar residues was bound per 500 base pairs in oxidized virus. One consequence of this in situ generation of formaldehyde is the formation of DNA-protein crosslinks. The DNA so crosslinked showed different patterns of restriction fragments with endonucleases such as Hpa I, Hind III and Kpn I but not with Xho I.

Characterization of avian reovirus-induced cell fusion: the role of viral structural proteins.

Cell fusion induced by avian reovirus was analyzed using virus strain FC and Vero cells. One-step growth curves showed that cell fusion was directly associated with viral replication. Cell fusion occurred most efficiently at basic pH (8.0-8.5) and fusion from without could not be demonstrated. Actinomycin D, at low concentrations, increased cell fusion, and cycloheximide prevented cell fusion, indicating that viral protein(s) were responsible for the induction of cell fusion. Immunofluorescence tests indicated that viral proteins were present on the infected cell surface. Radioimmuno-precipitation identified structural proteins mu 2C and sigma 2 as predominant viral protein species present on the infected cell surface. Cell fusion was inhibited by virus-specific antisera, suggesting that mu 2C and/or sigma 2 present on the infected cell surface were involved in the induction of cell fusion. Trypsin and chymotrypsin treatment of purified viruses cleaved both mu 2C and sigma 2 proteins, but generated different cleavage products with each protein. The addition of trypsin to the culture media following infection increased cell fusion, whereas chymotrypsin treatment decreased cell fusion. The opposite effects of trypsin and chymotrypsin on the cell fusion, together with the different specificities of these two proteases in cleavage of mu 2C and sigma 2 proteins, further suggest that the cell surface-associated mu 2C and/or sigma 2 are involved in the syncytium formation.