Biomedical Innovations to Combat COVID-19

By Sergio Rosales-Mendoza

Biomedical Innovations to Combat COVID-19 provides an updated overview on the development of vaccines, antiviral drugs and nanomaterials, and diagnostic methods for the fight against COVID-19. Perspectives on such technologies are identified, discussed, and enriched with figures for easy understanding and applicability. Furthermore, it contains basic aspects of virology, immunology, and antiviral drugs that are needed to fully appreciate these innovations.

This book is split into four sections: introduction, presenting basic virologic and epidemiological aspects of COVID-19; vaccines against COVID-19, discussing their different types and applications used to develop them; diagnostic approaches for SARS-CoV-2, encompassing advanced sensing and microfluidic-based biosensors; and drug development and delivery, where antivirals based on nanomaterials or drugs are presented.

It is a valuable source for virologists, biotechnologists, and members of biomedical field interested in learning more about how novel technologies can be applied to fasten the eradication of the COVID-19 and similar pandemics.

• Presents updated literature coverage summarizing the most relevant information on COVID-19
• Written by experts from diverse scientific domains in order to provide readers with a thorough view on the subject
• Encompasses tables, figures and information trees especially developed for the book in order to condense and highlight key points for quick reference

• Language: English
• Publisher: Academic Press
• Release date: Oct 15, 2021
• ISBN: 9780323902496

Book preview

Chapter 1

Basic virological aspects of SARS-CoV-2

M. Comas-Garcia¹,²,³, E.I. Rubio-Hernández⁴, I. Lara-Hernández², M. Colunga-Saucedo³, C.G. Castillo⁴, A. Comas-Garcia³,⁵, A. Monsivais-Urenda⁶,⁷ and R. Zandi⁸,    ¹Department of Sciences, Autonomous University of San Luis Potosí, San Luis Potosí, Mexico,    ²High-Resolution Microscopy Section, Research Center for Health Sciences and Biomedicine, Autonomous University of San Luis Potosí, San Luis Potosí, Mexico,    ³Medical Genomics Section, CICSaB, UASLP, San Luis Potosí, Mexico,    ⁴Coordination for the Innovation and Applicattion of Science and Technology (CIACYT)—Medical School, UASLP, San Luis Potosí, Mexico,    ⁵Department of Microbiology, Medical School, UASLP, San Luis Potosí, Mexico,    ⁶Translational Medicine Section, CICSaB, UASLP, San Luis Potosí, Mexico,    ⁷Department of Immunology, Medical School UASLP, San Luis Potosí, Mexico,    ⁸Department of Physics and Astronomy, University of California, Riverside, CA, United States

Abstract

Coronaviruses (CoVs) belong to a group of positive-sense single-stranded RNA viruses that have the largest genome known for a virus and use RNA as its genetic material. These viruses can infect a wide variety of animals, causing very different diseases that include common cold (humans), peritonitis (cats), hepatitis (mouse), and life-threatening pneumonia (humans). In fact, before the year 2002, most of the CoV literature focused on viruses of veterinarian interest. This changed in 2002 and 2012 with the appearance of two novel human CoVs that cause Severe Acute Respiratory Syndromes, SARS-CoV and MERS-CoV, respectively. The localized epidemics caused by these two viruses served as warning events on how zoonotic transmission of CoVs between bats (or camels) and humans could result in the formation of new viruses. Unfortunately, the research on these viruses mostly caught only the attention of the groups that either previously worked with CoVs of veterinarian interest or lived in the regions affected by SARS-CoV and MERS-CoV.

Keywords

SARS-CoV-2; coronaviruses; infectious cycle; virology; positive-sense single-stranded RNA viruses

1.1 Introduction

Coronaviruses (CoVs) belong to a group of positive-sense single-stranded RNA viruses that have the largest genome known for a virus and use RNA as its genetic material. These viruses can infect a wide variety of animals, causing very different diseases that include common cold (humans), peritonitis (cats), hepatitis (mouse), and life-threatening pneumonia (humans). In fact, before the year 2002, most of CoV literature focused on viruses of veterinarian interest. This changed in 2002 and 2012 with the appearance of two novel human CoVs that cause Severe Acute Respiratory Syndromes, SARS-CoV and MERS-CoV, respectively. The localized epidemics caused by these two viruses served as warning events on how zoonotic transmission of CoVs between bats (or camels) and humans could result in the formation of new viruses. Unfortunately, the research on these viruses mostly caught only the attention of the groups that either previously worked with CoVs of veterinarian interest or lived in the regions affected by SARS-CoV and MERS-CoV.

The emergence of a novel human CoV (SARS-CoV-2) in Wuhan, China at the end of 2019 changed the world. By the first trimester of 2020, this virus was almost in every continent, causing an unprecedented halt of social, academic, economic, cultural, sports, and even political activities. This pandemic not only changed the way we behave but also the way science is done. Since the release of the first full-length SARS-CoV-2 genome, in February of 2020 by the group of Prof. Zhang (Wu et al., 2020), research groups from all around the world decided to learn about CoVs and develop new research interests. Most importantly, in order to fight this pandemic, we need to develop new biomedical and biotechnological tools to understand, at least at a basic level, how CoVs (especially SARS-CoV-2) function.

The goal of this chapter is to establish the minimal virological basis required to understand the viral infectious cycle. It is not our intention to provide a detailed review of every single aspect of the viral infectious cycle but to highlight key parts of it. In fact, we apologize to everyone whose work has not been highlighted. This chapter is intended to help the reader navigate through the rest of the book and to appreciate the novel biotechnological approaches that have been developed to fight the COVID-19 pandemic. This chapter presents the different steps of the viral infectious cycle as it occurs in the cell. First, we introduce the virus by giving a description of the genome organization and the function of some of its genes; afterward, we address virion entry, replication, assembly, and egress. Finally, we added a section regarding the immune response to a viral infection. The goal of this section is to provide the readers with the minimal knowledge required to understand and appreciate the rest of the chapters.

1.2 Genome organization and function

CoVs belong to the order Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae, which are further divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (V’kovski et al., 2020). Alphacoronavirus and Betacoronavirus are the only CoVs known to infect mammals. Their genome is a monocatenary single-stranded RNA [(+)ssRNA] polycistronic molecule that is 5′-end capped (⁷mGpppN and ⁷mGpppm2N) and 3′-end polyadenylated. The length of the CoVs genome can range between 27,318 (HCoV-293) and 31,356 nucleotides (nt) [mouse hepatitis virus strain A59 (MHV-A59)] (Brian & Baric, 2005); the length of SARS-CoV-2 isolates uploaded on the NCBI database ranges from 29,903 (reference sequence NC_045512.2) (Wu et al., 2020) to 30,119 nt (MT844089) (Dinnon et al., 2020).

1.2.1 Genome organization

The genome organization in CoVs is as follows: the first two-thirds encode for all the nonstructural proteins that are required for genome replication and transcription (for a definition of these two processes, please go to the Section 1.4). The last third of the genome contains multiple overlapping open reading frames (ORFs) from where the structural and accessory proteins are translated. The structural genes are an absolute requirement for the viral infectious cycle, while the requirement of accessory proteins depends on the host.

As noted above, these viruses have the largest (+)ssRNA genome known to date; CoVs are the only ssRNA viruses that encode for an exonuclease and an endonuclease. These enzymes are responsible for the RNA-dependent RNA polymerase (RdRp) proofreading mechanism; therefore, they are important for viral replication and fitness. These enzymes stabilize their genome, allowing them to have such a large monocatenary genome.

The clinical isolates from the COVID-19 epidemic in Wuhan, China, were identified to belong to the Betacoronavirus genera. These isolates were a new virus, rather than a new strain of SARS-CoV, with a 91.02% and 90.55% identity to Pangolin-CoV and BatCoV RaTG13 at the whole genome level, respectively (Zhang et al., 2020). Perhaps the degree of identity between SARS-CoV-2 and these two viruses is not surprising; the percentage of identity between Pangolin-CoV (MT12126) and BatCoV RaTG13 (MN996532) is 90.05%.

While the origin of SARS-CoV-2 is still highly debatable, one of the current hypotheses is that bats are the probable reservoir host and that the Malayan pangolins (Manis javanica) could have been the intermediate species (Lam et al., 2020). This hypothesis is partially based on the fact that five critical residues of the receptor-binding domain (RBD) of SARS-CoV-2 are 100% identical to Pangolin-CoV, while RaTG13 shares only one amino acid in this region (Lam et al., 2020). Nonetheless, given the small number of known CoVs, the origin of SARS-CoV-2 is still an unanswered question.

It should be mentioned that most protein functions and interactions of SARS-CoV-2 are predicted by sequence and structure protein homology with other viruses (e.g., SARS-CoV and MHV). The role of these proteins in the viral infectious cycle will be explained in more detail in the following sections.

Most CoVs genomes contain around 10 ORFs. However, the exact number of ORFs and proteins depend on the viral species. As seen in Fig. 1.1, all the nonstructural genes are located in the OFR1, which has two ORFs: ORF1a and ORF1b. The latter is generated by a −1 ribosomal frameshifting. The last third of the genome codes for the four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Also, there are several accessory genes interspersed (or that overlap) between the structural genes. The expression of the structural and accessory proteins depend on the synthesis of "nested subgenomic RNAs." This mechanism will be explained in the Section 1.4. It is important to point out that the number and function of these accessory genes depend on the virus. Their functions might be related to host pathogenicity, given that some of them do not appear to be necessary for viral replication in cultured cells (Enjuanes et al., 2006; V’kovski et al., 2020).

Figure 1.1 Genome organization of SARS-CoV-2. (A) The pp1a and pp1b are synthesized from mRNA1 (gRNA) and then are proteolytically processed by PL and 3CL proteases into 16 non-structural proteins. B) The structural proteins (S, E, M, and N) and the accessory proteins 3a, 6, 7a, 7b, and 8 are translated from nested subgenomic RNAs. Protein 9b is generated by a -1 ribosomal frame shift from the same mRNA as N. The existence of 10 is controversial, also the presence of proteins 3b, 3c, and 3d are predicted by homology to SARS-CoV, but have not been detected yet. This scheme is based on the data of Kim et al. (2020) who indicate that the ORF7a and 7b are made from individual subgenomic RNAs.

1.2.2 Genome function

The ORF1a and ORF1b produce two large polyproteins: pp1a and pp1ab. The ORF1a contains 10 multifunctional nonstructural proteins (nsp1–nsp10) and ORF1b contains 6 (nsp11–nsp16). Most of these nsps have been studied in other CoVs and based on their sequence homology have been used as a reference to assign functions to SARS-CoV-2 nsps (V’Kovski et al., 2019) (Table 1.1). Except for nsp1, the rest of the 16 nsps encoded in the ORF1a and 1b compose the replication and transcription complex (RTC) (Gorbalenya et al., 2006). This complex interacts with endomembranes from the host endoplasmic reticulum and includes multifunctional viral proteins (Manfredonia et al., 2020) (Table 1.1). The primary role of these proteins is to replicate the viral genome and to transcribe the subgenomic RNAs (sgRNAs). As a consequence of the replication of the viral RNA, these proteins cause a large degree of cell remodeling, which will be discussed in the following sections (Goldsmith et al., 2004; Klein et al., 2020; Knoops et al., 2008; Stertz et al., 2007). However, some of them might have some effect on the host cell cycle (e.g., nsp1) or might disrupt intracellular signaling (e.g., nsp2). In comparison with other CoVs, the SARS-CoV-2 nsp3 has a large insertion between its two putative functional domains; these are homologous to the N-terminal domain (NTP) and the ADRP (or Mac-1) of SARS-CoV (Srinivasan et al., 2020).

Table 1.1

Most these proteins are related to the replication/transcription process, although some can participate in inhibiting the immune response.

The structural proteins are essential for virion assembly, entry to the host cell, and egress of viral progeny (Table 1.2). The spike protein (S) is highly glycosylated and participates in receptor binding and cell entry; therefore, it is essential in determining tropism. This protein forms homotrimers and has an unusually flexible linker that joins the ectodomain with the transmembrane and cytoplasmic domains (Strollo & Pozzilli, 2020; Walls et al., 2020). It is found in all CoVs, as will be discussed later on. Nonetheless, different CoVs proteins bind to different receptors; SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (hACE2) using the RBD. It can bind to TMPRSS2 (Hoffmann, Kleine-Weber, & Pohlmann, 2020; Hoffmann, Kleine-Weber, & Schroeder et al., 2020; Zang et al., 2020). In contrast, MERS-CoV, which is also a Betacoronavirus, binds to dipeptidyl peptidase 4 (dpp4, or CD26) (Park et al., 2019). It has been suggested that SARS-CoV-2 can employ dpp4 as coreceptor (Strollo & Pozzilli, 2020).

Table 1.2

The membrane protein (M) is an integral membrane glycoprotein with an ectodomain (N-terminal), three consecutive helical transmembrane domains (connected by two intravirion domains), and a long cytoplasmic domain (C-terminal) that interacts with the S protein (De Haan et al., 1998). It is one of the key proteins involved in virion assembly; it interacts with N, E, and S (Siu et al., 2008).

The envelope protein (E) is also an integral membrane glycoprotein and with a helical domain that connects the ectodomain (N-terminal) with the intravirion domain (C-terminal) (Maeda et al., 2001). While the concentration of this protein in the virion is lower than the other structural proteins, it is important for virion assembly and release, as well as in viral pathogenesis (Ruch & Machamer, 2012; Siu et al., 2008; Teoh et al., 2010).

The nucleocapsid protein (N) contains two highly structured domains, connected by a highly disordered linker. The NTD interacts with the viral RNA, while the C-terminal domains (CTDs) are responsible for protein–protein interactions, which bind to the viral RNA(Siu et al., 2008). Furthermore, it interacts with the nsp3 in the RTC (Cong et al., 2020); therefore, it plays a role in CoV RNA synthesis (Zuniga et al., 2010). Finally, it has been suggested that N has a role in fighting the host cell immune response by acting as an interferon antagonist (Almazán et al., 2004).

There are at least seven ORFs interspersed with the structural proteins that have been reported for SARS-CoV-2: ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF9b, and ORF10 (although the expression of protein 10 is controversial). Four accessory proteins present in most CoVs have not been found in SARS-CoV-2-infected cells. Therefore their existence is predicted based on their sequence homology with other CoV proteins with known functions. The functions of these proteins are summarized in Table 1.3 (most of the data in this table were obtained from https://covid-19.uniprot.org).

Table 1.3

Most functions of the accessory proteins are assumed by their sequence homology to SARS-CoV proteins.

The 3a protein is a viroporin (ion channel) that disrupts acidification of the lysosome and will be carefully explained in Section 1.6. This protein plays an important role in viral egress by deacidification of the lysosomal egress pathway (Castaño-Rodriguez et al., 2018; Ghosh et al., 2020; Lu et al., 2006; Yue et al., 2018). It has been proposed that, in addition to N, proteins 3b and 6 are interferon antagonists (Konno et al., 2020; Kopecky-Bromberg et al., 2007; Yue et al., 2018). Jiang et al. proposed that there is an ORF9b that is expressed by leaky scanning of the N gRNA and acts as an interferon antagonist (Jiang et al., 2020). Bioinformatic analysis predicts that the SARS-CoV-2 genome can be recognized by the Toll-like receptors 7/8, which are part of the innate immune system and produce type I interferon; therefore, protein 9b could be one of the keys for the evasion of the innate immune system (Moreno-Eutimio et al., 2020). Furthermore, there is another hypothetical 38-amino-acid-long protein downstream of the N gene (ORF10). This gene product has been found as prematurely terminated protein in infected patients. However, some reports have questioned the presence of the protein 10 in infected cells (Bojkova et al., 2020; Davidson et al., 2020; Kim et al., 2020). In fact, it is important to mention that not all accessory proteins have been detected in infected cells (Davidson et al., 2020; Kim et al., 2020).

The differences in the accessory proteins between SARS-CoV and SARS-CoV-2 are not only in the number of proteins or their functions but also in their lengths. For example, the SARS-CoV-2 ORF3b has a premature codon not present in its SARS-CoV counterpart. The SARS-CoV-2 ORF3b has not yet been detected in infected cells (Bojkova et al., 2020; Davidson et al., 2020). Moreover, during the SARS-CoV pandemic, there was a deletion of the ORF8 (He, Peng, et al., 2004; He, Zhou, et al., 2004; Muth et al., 2018), which has not been observed yet for SARS-CoV-2 except for one variant detected in Singapore (Su et al., 2020). This deletion is very surprising since it is likely that protein 8b mediates degradation of the major histocompatibility complex (Park, 2020; Park et al., 2019).

Finally, while most protein databases and articles assign several functions to the SARS-CoV-2 nonstructural, structural, and accessory proteins, there is yet a long way to determine if the predicted functions, by homology with other CoVs (especially with SARS-CoV), are valid. It is possible that some of these accessory proteins have functions that are completely different from those of closely related CoVs. Therefore it is of key importance to determine their role (in vitro and in vivo) in pathogenesis, infectious cycle, and viral fitness.

In summary, the genome of SARS-CoV-2 is a capped and polyadenylated single-stranded RNA molecule that contains multiple ORFs. The ORF1 is translated from the genomic RNA and produces the polyproteins pp1a and pp1ab. These polyproteins are processed by cellular and viral proteases to produce 16 nonstructural proteins. The function of most of these proteins is mainly related to replication and transcription of the viral genomic and messenger RNAs. However, some of them can participate in evading the host-cell immune response or even in viral egress. The structural and accessory proteins are translated from nested subgenomic RNAs. The structural proteins are defined as those that are required for virion assembly; however, they can have multiple functions. They can participate in the inhibition of the host-cell immune response or transcription/replication. The accessory proteins are not essential for assembly or replication, but they are key for viral pathogenesis. In particular, they participate in host modulation by inhibiting interferon response, deacidification of the lysosome, and dysregulation of the cell cycle. The exact number and function of the accessory proteins are still investigated. In fact, the presence and function of most of the accessory proteins have been assigned based on their sequence homology to other CoVs. Further studies might reveal that these genes could have novel or unique functions compared to SARS-CoV or other human CoVs.

1.3 Viral entry

The first step in the viral infectious cycle is the attachment of the virion to the host–cell plasma membrane; this is the result of interactions between the viral particle and the host-cell receptor. For enveloped viruses, such as CoVs, the lipid bilayer that surrounds the nucleocapsid is decorated with multiple copies of at least one viral transmembrane glycoprotein. These proteins are responsible for virus–cell interaction and tropism. In some cases, the interaction between the glycoproteins and the cell receptor can be sufficient for viral entry. However, in viruses, such as CoVs, entry is a complex process and requires other host–cell molecules (coreceptors and/or attachment factors) that include proteases or sialic acids. If a virus requires a coreceptor, the expression of the receptor is necessary but not sufficient for entry. Therefore the susceptibility of any given cell type to be infected by a particular virus is the sum of all the interactions required for the viral particle to bind to the cell membrane, cross it, and deliver its genome to the right place. Using the wrong entry mechanism could lead to the viral genome not being released in the proper place; therefore, not being able to initiate the viral infectious cycle.

As noted above, a CoV virion consists of a ribonucleoprotein complex (RNP) surrounded by a viral membrane decorated with an integral membrane protein (M), a small membrane protein (E), and a transmembrane spike glycoprotein (S) (Fig. 1.2). The protein N interacts with the genomic RNA to assemble the ribonucleic complex. The interaction of this complex with the M protein results in the packaging of the genomic RNA in the virion (Escors et al., 2001; Fehr & Perlman, 2015; Narayanan et al., 2000). Moreover, there is evidence that M interacts with S (Fehr & Perlman, 2015; Mortola & Roy, 2004; Opstelten et al., 1995). The small membrane protein E has an ion channel activity and is required for assembly (Ruch & Machamer, 2012) and membrane remodeling (Wilson et al., 2004). Furthermore, it plays a role in the egress of the virus from the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) (Ruch & Machamer, 2012). Although it might remodel other membranes. On the one hand, M and E are required for assembly (Huang et al., 2004; Tseng et al., 2010), although expression of N increases the yield of assembly (Siu et al., 2008). On the other hand, from the perspective of viral entry, the most important protein is S. The protein S interacts with the cellular receptor and determines tropism (see Table 1.4 for different CoVs, receptors, and tropism).

Figure 1.2 Architecture of the SARS-CoV-2 virion.

The cryo-electron microscopy reconstruction of a viral particle shows that within the same virion, there are two types of trimeric S protein. Some trimers have one receptor-binding domain (RBD) in the up conformation (red) and some have all of them in down conformation (salmon). The RBD in the up conformation establishes interactions with the host-cell receptor. Source: Modified from Yao, H., Song, Y., Chen, Y., Wu, N., Xu, J., Sun, C., Zhang, J., Weng, T., Zhang, Z., Wu, Z., Cheng, L., Shi, D., Lu, X., Lei, J., Crispin, M., Shi, Y., Li, L., & Li, S. (2020). Molecular architecture of the SARS-CoV-2 virus. Cell, 183(3), 730–738. https://doi.org/10.1016/j.cell.2020.09.018.

Table 1.4

Suffixes h, c, and f refer to human, canine, and feline species. CCoV, Canine CoV; FeCoV, feline CoV; PEDV, porcine epidemic diarrhea virus; MHV, murine hepatitis virus.

1.3.1 Virus–cell interaction

The interaction between the host–cell receptor and the protein S from different CoVs has been widely documented (Table 1.4). The receptor for CoV-NL63 (Hofmann et al., 2005), SARS-CoV (He, Peng, et al., 2004; Muth et al., 2018), and SARS-CoV-2 (Jian et al., 2020; Wang et al., 2020) is the hACE2. Interestingly, CoV-NL63 is an Alphacoronavirus and yet it uses the same cell receptor as SARS-CoV and SARS-CoV-2. The interaction of the hACE2 receptor and SARS-CoV (Li, 2015) and SARS-CoV-2S proteins is mediated by the RBD (Hoffmann, Kleine-Weber, & Pohlmann, 2020; Zang et al., 2020; Ou et al., 2020). This domain is constantly alternating between two different conformations: a "standing-up position in which it interacts with the receptor and a lying-down" conformation allowing the virus to evade the immune system (Yuan et al., 2020) (Fig. 1.3). It is important to point out that the standing-up conformation in the trimeric presentation of S occurs for only one of the three RBDs, the other two domains remain in the lying-down conformation (Walls et al., 2020). It is possible that due to steric repulsion only one RBD can be in the standing-up position.

Figure 1.3 Cryo-electron microscopy structure of the conformation of the trimeric S protein from SARS-CoV-2. (A–C) Different views of the closed conformation in which all the receptor-binding domains (RBDs) are lying-down. (D–F) Different views of the partially opened trimer; one of the three RBDs are in the stand-up conformation. Source: From Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281–292. https://doi.org/10.1016/j.cell.2020.02.058

Yang et al. compared the RBDs from SARS-CoV and SARS-CoV-2 and found that 75% of the amino acids are conserved (Yang et al., 2020). Furthermore, only 57% of the amino acids identified as important for receptor binding are conserved between both viruses. Therefore it is not surprising that there are some structural and functional differences between the RBDs of SARS-CoV and SARS-CoV-2. In reality, the binding mechanism is not as simple as it might appear. On the one hand, the SARS-CoV-2 RBD has a higher affinity for hACE2 than the SARS-CoV RBD does. On the other hand, the affinity of the full-length S protein of SARS-CoV-2 for hACE2 is comparable or lower that of SARS-CoV for the hACE2 (Jian et al., 2020; Walls et al., 2020) (Fig. 1.4). These results show that the entry process should not be reduced to the interaction of a portion of S and the receptor, but it has to take into account the full-length S protein.

Figure 1.4 Binding assays of the SARS-CoV-2 and SARS-CoV S protein to hACE2. Binding assays of the SARS-CoV-2 (A) and SARS-CoV (B) recombinant S protein to hACE2 measured by biolayer interferometry. The dotted lines correspond to using a 1:1 binding isotherm. The dissociation constants (KD) for SARS-CoV-2/hACE2 and SARS-CoV/hACE2 are 1.2 and 5.0 nM, respectively. This difference is not enough to determine a difference in the in vivo binding affinity to the hACE2 receptor. Source: From Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281–292. https://doi.org/10.1016/j.cell.2020.02.058.

There is evidence that SARS-CoV-2 can also use the transmembrane receptor neurophilin-1 (NRP1) (Daly et al., 2020). This receptor is highly expressed in the respiratory and olfactory epithelium; therefore, it might allow infection of the central nervous system (CNS). Davies et al. demonstrated that NRP1 is expressed in the CNS, including olfactory-related regions (Davies et al., 2020). This could explain reports on neurologic disorders in hospitalized COVID-19 patients (Collantes et al., 2020; Frontera et al., 2020; Varatharaj et al., 2020). The facts that this virus can use very different cellular receptors and that the disease it causes results in multiorgan damage indicate that it can replicate in different cell types; therefore, there might be more receptors involved in the viral entry

Besides the small differences in the binding affinities between the SARS-CoV and SARS-CoV-2 RBDs to the hACE2 there are other aspects in the viral entry process that result in important biological differences. First, the S protein of SARS-CoV-2 has a four amino acids insertion (RRAR) at the S1/S2 boundary that results in the presence of a furin cleavage site (Hoffmann, Kleine-Weber, & Pohlmann, 2020; Walls et al., 2020; Wrapp et al., 2020) (Fig. 1.5), absent in SARS-CoV (Hoffmann, Kleine-Weber, & Pohlmann, 2020). A similar basic motif is present in CoV-OC43, MERS-CoV, and CoV-HKU1 (Fig. 1.6). In MERS-CoV, the S protein is activated by a two-step process that requires furin and TMPRSS2 (Hoffmann, Kleine-Weber, & Pohlmann, 2020). Hoffman et al. have shown that the insertion of the RRAR motif into the SARS-CoV S protein results in a protease-dependent viral entry (Hoffmann, Kleine-Weber, & Pohlmann, 2020). Based on these results, they suggested that furin processing of the S protein, either during egress or at the surface of the target cell, is key for viral entry.

Figure 1.5 Structure of SARS-CoV-2S in the prefusion conformation. (A) Schematic of the SARS-CoV-2S protein primary structure. SS, signal sequence; S2′, S2′ protease cleavage site; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain; and CT, cytoplasmic tail. Indicate the furin cleavage sites. (B–C) Two orthogonal views of the trimeric S protein with one RNA in the standing-up conformation. Source: From Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., Graham, B. S., & McLellan, J. S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. science. Science, 367(6483), 1260–1263. https://doi.org/10.1126/science.abb2507.

Figure 1.6 The sequence alignment of residues around S1/S2 and the S2′ cleavage for multiple CoVs. The sequence alignment of residues around S1/S2 and S2′ cleavage sites show that the RRAR furin cleavage site is unique among human and most bat CoVs. Source: Modified from Hoffmann, M., Kleine-Weber, H., & Pohlmann, S. (2020). A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular Cell, 78(4), 779–784. https://doi.org/10.1016/j.molcel.2020.04.022.

Shang and coworkers used biochemical and pseudovirus entry assays to investigate the role of the RRAR motif in the entry pathway of SARS-CoV-2 (Jian et al., 2020). Using a pseudotyped lentivirus, they showed that the S protein is cleaved at the RRAR motif during virus assembly, at least in HEK-293T cells. To show that the RRAR motif is required for S1/S2 cleavage, they replaced this sequence with the corresponding one from SARS-CoV; the mutant protein was not cleaved at the S1/S2 boundary site. This is in agreement with the findings of Hoffmann and coworkers who showed the same results but using a pseudotyped vesicular stomatitis virus (Hoffmann, Kleine-Weber, & Pohlmann, 2020). Insertion of the SARS-CoV-2 S1/S2 site into SARS-CoV resulted in increased cleavage of the S protein (Hoffmann, Kleine-Weber, & Pohlmann, 2020). Interestingly, they also showed that the insertion of the RaTG13 (a bat Betacoronavirus) S1/S2 into SARS did not have the same effect as it did for SARS-CoV-2. These observations imply that the basic character of the site is not enough for protease recognition and there is some sequence specificity

1.3.2 Clathrin-mediated endocytosis

The exact entry pathway of SARS-CoV-2 is currently being investigated; there is data supporting the hypothesis that this virus uses clathrin-mediated endocytosis (Jian et al., 2020). Nonetheless, the entry pathway for several CoVs has been determined. For example, although CoV-NL63 is an Alphacoronavirus, this virus and SARS-CoV-2 use the same main cell receptor. Therefore some aspects of the entry process could be conserved (Hofmann et al., 2005; Milewska et al., 2018). Mileswka et al. have studied the entry of CoV-NL63 to LLC-MK2 and HAE cells (Milewska et al., 2018). They observed that the binding of CoV-NL63 to hACE2 results in clathrin recruitment and formation of clathrin pits. In this study, no other markers were associated with the viral entry (e.g., caveolin). To determine if endosomes are required for CoV-NL63 entry to LLC-MK2 cells, they added ammonium chloride and bafilomycin A to the cell culture; virus entry was blocked in these experiments. They concluded that endosomes are needed for cell entry. Furthermore, virus internalization was blocked by inhibiting dynamin, a GTPase responsible for endocytosis. Interestingly, these endocytosis inhibitors did not block viral entry to HAE cells, suggesting that there could be an alternative entry pathway in this cell line. For example, the Betacoronavirus MHV-2, unlike other MHVs, does not require endosome acidification (Qiu et al., 2006). Moreover, MHV-4 can enter through endosomal and nonendosomal pathways (De Haan et al., 2006; Nash & Buchmeier, 1997). In fact, there is evidence that the S protein of this CoV S proteins is processed by TMPRSS2 and thus may use an alternative entry route in HAE cells (Schickli et al., 1997). This observation is of special interest for SARS-CoV-2 since the entry requires priming by TMPTSS2 (Hoffmann, Kleine-Weber, Schroeder, et al., 2020). Finally, by using acting polymerization inhibitors, jasplakoline, and cytochalasin, Mileswka et al. (2018) showed that the internalization of CoV-NL63 requires active actin polymerization. Ou et al. showed that the SARS-CoV-2 entry requires processing by lysosomal proteases. Therefore its entry may require the fusion of the endosome with lysosome.

Shang et al. have also shown that the inhibition of this processing step resulted in a decrease in the infection of HeLa/hACE2, Calu-3, and MRC-5 cells (Jian et al., 2020). Furthermore, treating the producer cells with PCC inhibitors (PCCi) resulted in a decrease in infectivity of the pseudotyped virus. As expected, the treatment of SARS-CoV producing cells with these inhibitors did not affect infectivity. Interestingly, there was some residual entry that was attributed to the activity of other proteases. However, based on the data from Shang et al. (Jian et al., 2020) and (Milewska et al., 2018), it is possible that pseudotyped viruses with the mutant SARS-CoV-2S protein could enter through an alternative route (albeit being less efficient than the canonical one). This hypothesis is further reinforced by experiments in which SARS-CoV-2 entry is reduced by inhibition of TMPRSS2 with camostat or lysosomal cathepsin (Jian et al., 2020). An interesting observation is that a combination of PCCi and camostat or E64d reduced entry, but did not completely block it; the combination of inhibitors decreased the relative entry efficiency to about 25%. Furthermore, the combination of PCCi and camostat did not affect the entry of the SARS-CoV S pseudotyped virus, while either E64d or E64d plus PCCi almost completely inhibited this virus, indicating that only E64d inhibits SARS-CoV entry. These data show that inhibition of lysosomal cathepsins is enough to block entry of SARS-CoV-2 but not SARS-CoV.

In summary, the data presented here results in a scenario where entry of SARS-CoV-2 requires priming of the S protein by the cellular protease furin. This process is a conformational activation of the S protein by a furin-mediated cleavage of the amino acids at the boundary of the S1 and S2 domains. Priming of the S protein requires the amino acid motif RRAR, which is not present in SARS-CoV. After the virion binds to the cell-receptor (hACE2), the viral particle enters by clathrin-mediated endocytosis. The SARS-CoV-2 replication cycle utilizes an endocytic pathway that involves endosome/lysosome fusion. Inhibition of this fusion process decreases viral infectivity. Therefore the current model indicates that the processing of SARS-CoV-2 by lysosomal proteases is a required step of the viral cycle. The infection of SARS-CoV does not depend on functional lysosomal proteases. All these show that there is still a great deal of work to be done. For example, the number and identity of alternative cell receptors and coreceptors are yet to be determined. The mechanism by which the lysosomal proteases contribute to the infectious cycle, or how the viral RNP escapes the lysosome, is not clear.

1.4 Genome replication and translation

The viral replication of some (+)ssRNA viruses (e.g., alphavirus and coronavirus) requires the synthesis of two different types of RNAs: a genomic RNA (gRNA) and one or more subgenomic RNAs (sgRNAs). The former refers to the full-length positive-sense RNA molecule that codes for the complete genetic information of the virus (which is also an mRNA), while the latter are shorter versions of the gRNA that are used for the translation of the accessory and structural genes that are not accessible to the ribosomes in the full-length RNA. For these viruses, genome replication implies the synthesis of a full-length copy of the gRNA. This is done through a negative-sense RNA intermediate species (antigenome) (Fig. 1.7). However, transcription refers to the process by which the sgRNAs are synthesized. The transcription mechanisms of sgRNA are very diverse; therefore, we will focus only on the mechanism used by CoVs. When we refer to CoV transcription, we mean the synthesis of sgRNAs from a (−)ssRNA intermediate that is shorter than the full-length antigenome.

Figure 1.7 Replication of the SARS-CoV-2 genomic RNA. The positive sense (+) genomic RNA is used as template for the synthesis of a (−) full-length RNA intermediate. The RNA-dependent RNA polymerase (RdRp) that forms part of the replication/transcription complex (RTC) binds to the 3′-end of the gRNA to synthesize a full-length complimentary copy of the antigenome; afterward, the RdRp binds to the 3′-end of the (−) RNA to make a full-length size complimentary copy of the template RNA to produce a new (+) gRNA. Moreover, the gRNA can be used as template for the synthesis of the antigenome. The leader and body TRS are in blue and hot pink, respectively. The anti-TRS-L and anti-TRS-B in striped blue and pink, respectively. The genomic RNA is 5′-end capped and 3′-end polyadenylated. The ORF1b is read by a −1 ribosomal frameshift. It is important to mention that the existence of the ORF10 is in question. The striped regions correspond to the complementary sequences of the gRNA. For illustrative reasons, the scale of the genome is not linear and the relative position of each gene is indicated by the upper scale.

1.4.1 Replication and transcription

The synthesis of viral RNA is performed by nsp12 RpRp and its cofactors nsp7 and nsp8 (Table 1.1), the integrity of the synthesized RNA is maintained by nsp14, and the mRNAs are 5′-end capped (⁷mGpppN and ⁷mGpppm2N) by nsp10, nsp13, nsp14, and nsp15 (V’kovski et al., 2020).

As mentioned in the first section, the canonical translation ORF1a produces pp1a, while a −1 ribosomal shift produces pp1ab that results in the addition of six more proteins (V’kovski et al., 2020). For SARS-CoV-2, the efficiency of this ribosomal frameshift is around 57% (Finkel et al., 2021); this process depends on a conformational change of the secondary structure of an RNA element between nucleotides 13,408 and 13,492 (Lan et al., 2020). At any rate, these two polyproteins are synthesized from the same mRNA: the gRNA and mRNA1. This mechanism is common amongst (+)ssRNA viruses (e.g., Coronaviridae, Astroviridae, and Togaviridae) and it is even present in retroviruses. Furthermore, CoVs use the discontinuous synthesis of nested sgRNAs to generate mRNAs for the structural and accessory genes (Sawicki et al., 2007; Sawicki & Sawicki, 1998) (Fig. 1.8). This is characteristic of viruses of the order Nidovirales, although the exact mechanism might vary between viral families.

Figure 1.8 Transcription of the CoV sgRNAs by discontinuous synthesis. The gRNA has a stop codon at the end of the ORF1a; however, there is a −1 ribosomal frameshift that allows for the synthesis of an extended polyprotein (pp1ab). The synthesis of the sgRNAs (mRNA2–10) occurs by a discontinuous synthesis of a (−) sgRNA intermediate molecule by the RNA-dependent RNA polymerase (RdRp) that is present in the replication and transcription complex (RTC). The RdRp binds to the 3′-end of the gRNA (template) and creates a complimentary copy. However, instead of doing a full-length copy, the RdRp stops at the TRS-B (in this case the one for the S gene); afterward, the RdRp/(−)sgRNA complex jumps to the 5′-end, where it binds to the TRS-L to continue with the rest of the synthesis. The (−)sgRNA is used as template for the synthesis of the sgRNAs, which are mRNAs for the structural and accessory proteins. In this case, we have represented the synthesis of the mRNA2 that is used for the translation of S. The gRNA and sgRNA are 5′- and 3′-end capped and polyadenylated, respectively. The green regions indicate each of the genes (ORF3a and 9 can produce other proteins by leaky scanning). The light green region indicates that the ORF1b is translated by a −1 ribosomal shift. The stripped colors indicate the sequences complementary to those found in the gRNA. The dashed lines indicate base pairing between the anti-TRS-B and TRS-B, as well as between the anti-TRS-B and TRS-L. For the case of the mRNA, the green shading after the stop codon indicates that those genes cannot be translated from that mRNA. This mechanism results in mRNAs with the same 5′- and 3′-end sequences. For illustrative reasons the scale of the genome is not linear, the relative position of each gene is indicated by the upper scale.

The current model for the synthesis of nested sgRNAs was proposed by Sawicki and Sawicki (1998). In this model, the RdRp binds to the gRNA to produce shorter (−)sgRNAs; these (−)RNAs will be as used templates for each of the mRNAs2–mRNAs9 (possibly even a mRNA10 for SARS-CoV-2). It should be mentioned that is not clear how much the general mechanism proposed by Sawicki and Sawicki for CoVs differs from that of SARS-CoV-2. It is important to point out that this model is very different from the one used for many viruses such as those in the Togaviridae family (Rupp et al., 2015). For these viruses, the RdRp complex synthetizes a full-length (−)ssRNA (antigenome) that is used as a template for the synthesis of the gRNA and sgRNA. The gRNA is used for packaging and translation of the nonstructural proteins (ORF1), while the structural proteins (ORF2) are translated from the sgRNA. The synthesis of the sgRNAs occurs when the RdRp complex binds to the antigenome at a position that is located between the sequences that correspond to the intergenic region between ORF1 and ORF2 of the gRNA.

In general, this process requires two very important RNA regulatory elements. First, close to the 5′-end, there is a transcription regulatory sequence (TRS) adjacent to the leader sequence (TRS-L). Second, there is a set of TRSs elements located at the end of each ORF (except ORF1a/b) called body TRS or TRS-B. The 5′ proximal TRS-B determines the beginning of each of the sgRNAs; there are at least nine TRS-B. As it will be explained later on, the discontinuous synthesis of the sgRNA produces at least nine mRNAs; however, some of these mRNAs can act as a template for other proteins by using a leaky scanning mechanism.

As explained in Fig. 1.8, the discontinuous synthesis of the (−)sgRNAs leads to the generation of multiple templates that will be used for the transcription of each of the sgRNAs that encode for the structural and accessory proteins. The fusion of the leader-body TRS occurs during the synthesis of the negative strand (see dashed lines in Fig. 1.8). These transcriptional elements contain a short core sequence (CS) (ACGAAC) that is surrounded by variable sequences (Nomburg et al., 2020). There are few reports on the secondary structure of the SARS-CoV-2 genome (Huston et al., 2020; Lan et al., 2020; Manfredonia et al., 2020; Tavares et al., 2020). However, Lan et al. determined, by dimethyl sulfate mutational profiling sequencing (DMS-MaPseq) with a single-nucleotide resolution, the RNA secondary structure of the SARS-CoV-2 genome (Lan et al., 2020). This study revealed that not only the surrounding sequences of the CS of the TRS are variable but also their predicted secondary structures are very different from each other. It is important to point out that these noncanonical jumps can produce truncated