In early March 2020, the World Health Organization declared that the first pandemic caused by coronavirus disease 2019 (COVID-19). The COVID-19 infection sweeps the globe after the first report in Wuhan, China, in December 2019 that severe pneumonia was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By the beginning of March 2021, over 117 million people have been infected with SARS-CoV-2, and over 2.6 million correlated deaths have been reported worldwide (Worldometers.info, 2021).
The rapid progress of whole-genome sequence analysis revealed SARS-CoV-2 has a 29.9 kb-long, positive-sense single-stranded RNA as a genome and belongs to the genus β-coronavirus. SARS-CoV-2 shows approximately 79.5% of nucleotide similarity to SARS-CoV while having 96% homology to bat coronavirus (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020; Zhou
Coronaviruses utilize various membrane proteins on host cells as receptors, including aminopeptidase N for HCoV-229E and TGEV (Delmas
In humans, ACE2 is mainly expressed in lung epithelial cells, enterocytes, arterial endothelial cells, and smooth muscle cells in cardiovascular tissues (Hamming
This review aims to collate and organize the latest information on host proteins involved in the cell entry process. Scrutiny of the relationship between the host factors and viral proteins can help further understand of emerging SARS-CoV-2. Sophisticated information regarding invasive cellular traits can also provide an insight for designing antiviral drugs targeting virus-host protein interaction to perturb the virus entry.
The S protein is a trimeric glycoprotein embedded in the viral envelope and plays an essential role in viral binding to receptors. Because the S protein determines the tropism of viral infection, numerous efforts on functional as well as structural studies have been made, and its three-dimensional structure was quickly elucidated by cryo-EM analysis (Coutard
In the S2 region, an additional protein cleavage occurs to form S2′. This process also plays an essential role in determining the efficiency of viral infection to the target cells. Surprisingly, unlike SARS-CoV, the SARS-CoV-2 contains an additional protease recognition site in the S1/S2 cleavage area (Andersen
The S proteins of SARS-CoV and SARS-CoV-2 show approximately 76% genetic similarity. In contrast, the receptor-binding domain (RBD) in the S protein, responsible for binding to host receptors, sustains only a 50% similarity between two types of SARS-CoV. Of note, both SARS-CoV-2 and SARS-CoV utilize an identical type 1 membrane protein, ACE2, their primary cellular receptor (Wu
To bind ACE2, the RBD of S protein required the receptor-accessible conformation that shifts RBD conformation towards an ACE2 because of hidden and buried receptor-binding site (RBS) in the interspace of protomers (RBD closed) (Fig. 1). When the RBD conformation changes to OPEN state, RBD loses the interactions with S2 or other protomers, and RBS is exposed and then ready for the interaction with ACE2. Therefore, the two conformations of RBD, open and closed states, were related to viral attachment and infectivity (Walls
The binding of SARS-CoV-2 RBD to ACE2 involves 13 hydrogen bonds contributed by multiple tyrosine residues, two salt bridges, and the glycan at Asn-90 of ACE2, which is very similar to the binding of SARS-CoV to ACE2 (Yuan
Since SARS-CoV-2 uses RNA as a genome, continuous and sporadic nucleotide mutations throughout the viral genome accumulate during genome replication (Denison
Based on the identical mutation sites of E484K and N501Y retained in three different variant lineages localized in the receptor binding motif that binds to human ACE2. E484K substitution altered the hydrogen bonds or charged interactions that might effectively escape the neutralizing monoclonal antibodies, BD23, C119, and P2B-2F6. Moreover, E484K mutation decreased the neutralizing activity of vaccinated human sera with the Pfizer mRNA (BNT162b2) in a pseudotyped lentiviral virion system (Tada
Various host factors are involved during viral infection, including receptor(s), membranous proteases, and other cell surface proteins (Fig. 3). Herein, we will discuss the characteristics of the cellular receptor of SARS-CoV-2 and other host factors contributing to viral entry into the cells. Intrinsic functions, suggested roles of viral entry and inhibitors for each cellular protein are summarized in Table 1.
|Box 1. Currently known primary characteristics of the S protein of SARS-CoV-2 (Fig. 2)|
The S protein (~180 kDa) of SARS-CoV-2 consists of a trimeric glycoprotein structure in a viral envelope.
The S protein contains two domains, S1 and S2, and plays a critical role in cell entry binding to the host cell receptor, ACE2.
Open status of RBD in the S protein led to the exposure of the RBS (receptor binding site) and ready to interact with ACE2.
S protein binding to the ACE2 promotes viral envelope and cell membrane fusion and elicits structural changes.
Neuropilin-1 (NRP1) was suggested as a secondary cellular receptor factor that may facilitate virus entry into the cell.
RBD in the S protein is essential for recognizing receptors and generating neutralizing antibodies.
Notable mutations in the S protein were identified and retained at the receptor binding motif, which may neutralize or reduce currently available vaccines and antivirals.
The development of antiviral drug strategies to reduce viral entry has focused on the inhibition of interactions between the S protein and receptor machinery mediated by various host proteins.
Viruses are obligate intracellular parasites; thus, exploiting a host is essential for their reproduction. The first step of virus replication is to invade cells. The host cell surface proteins to which a virus binds are critical in determining the viral susceptibility of the host. Both SARS-CoV-2 and SARS-CoV are reported to use ACE2 as a receptor (Lan
ACE2 was identified as a receptor for the SARS-CoV, a causative agent of the SARS epidemic in 2003 (Li
The peptidase domain (PD) of ACE2 is the counterpart of the RBD in the S protein. The binding of PD to the RBD causes structural changes in the S protein, exposing the cleavage sites at the S1/S2 or adjacent regions, which are attacked by host cellular proteases, and prompting the conversion from pre-S to post-S states (Li
Given the role of ACE2 in regulating blood pressure, it is conceivable that hypertension could affect viral infection outcomes. For instance, animals administered with ACE inhibitors or angiotensin receptor blockers (ARBs) showed an increased expression of ACE2 in the lung and kidney tissues (Ferrario
However, the report of the enhanced expression of ACE2 after ACE inhibitors or ARBs treatment remains confirmed, and there is no evidence that similar treatments increase ACE2 expression in human lung tissues (Kuba
ACE2 is also found in a soluble form in blood and urine related to extracellular shedding of ACE2. A disintegrin and metalloproteinase 17 (ADAM17) is reported to be involved in this process (Wysocki
Sequence diversity and variable gene expression of ACE2 are well-documented among different populations (Nedelkov, 2008; Hussain
Although ACE2 is a primary rendezvous point on the cell for infection of SARS-CoV-2, it remains unclear whether the physiologic function of ACE2 impacts the infectivity of SARS-CoV-2. The signaling cascade downstream of ACE2 may be an alternative contributor to viral infectivity or productivity. It also remains to be clarified whether dysfunctional ACE2 contributes to damage inflicted on lung parenchymal cells and lung tissue, putting an additional disease burden on the host. Further elucidation for the precise role of ACE2 in viral infection could provide insight on how to strategize suppression of SARS-CoV-2 infection.
While most studies have focused on ACE2 as a unique cell receptor for viral entry, a novel cell entry facilitator, neuropilin-1 (NRP1), has recently been identified (Cantuti-Castelvetri
Unlike SARS-CoV, the S protein of SARS-CoV-2 possesses a furin cleavage site located adjacent to the S1/S2 junction containing an R-X-X-R motif, in which R and X represent glycine and any amino acid residue, respectively. An amino acid residue “R” is constantly exposed at the C-terminus of the S1/S2 cleavage site by the host protease furin, commonly known as the C-end rule (CendR) (Teesalu
Further investigation was conducted using the pseudovirus or the SARS-CoV-2 mutant lacking the furin recognition site to understand how viral infectious interference resulted in reciprocal interaction between the CendR motif of the S protein b1 domain by NRP1 (Cantuti-Castelvetri
However, several questions remain to be answered if NRP1 is considered a secondary cellular receptor in addition to ACE2, which has been initially shown to contribute to the determination of infectivity and transmissibility. First, unlike SARS-CoV, MERS-CoV contains a furin recognition site in its S protein. The underlying reason(s) why infectivity and transmissibility of MERS-CoV differ from SARS-CoV-2 requires further investigation. Second, it is necessary to elucidate how the furin-cleaved motif of S1 bound to the NRP1 b1 region is involved in viral cell entry using ACE2 as an essential prerequisite or an accessory structural behavior that assists in the S protein and ACE2 interaction. Finally, epidemiologic studies should be conducted on how the characteristics of NRP1, which are widely distributed in various tissues, especially within blood vessels, affect disease exacerbation of SARS-CoV-2 infected tissues as a secondary cellular receptor.
Compiling the appropriate knowledge on the importance and function of NRP1 within the infectivity and spread of the virus may implicate it as a meaningful target when developing antiviral drugs that inhibit the interaction between virus and cells.
When the S protein of SARS-CoV-2 is bound to ACE2, fusion mediated by host proteases ensues from the viral envelope and cell membrane. Host proteases mediate this process, and a series of structural changes in the S protein subsequently occur, including 1) cleavage at the S1/S2 region of the viral S protein, 2) breakage at the S2′ in S2, which results in the exposure of the fusion peptide, and 3) the formation of the heptad structure of heptad repeat 1 (HR1) and 2 (HR2) (Xia
No correlation was established between the expression levels of ACE2 and infectivity of SARS-CoV in 2003 (Ding
Indeed, SARS-CoV-2, unlike SARS-CoV or other β-coronaviruses, contains a unique proteolytic region near the S1/S2 cleavage site, which can be recognized by other cellular proteases rather than TMPRSS2 through viral genomic analysis (Coutard
The S protein in SARS-CoV-2 harbors a furin cleavage site in the S1/S2 region, which does not exist in SARS-CoV (Kleine-Weber
Furin recognizes and cleaves the R-X-X-R motif in a calcium-dependent manner where R and X represent glycine and any amino acid residues, respectively. (Nakayama, 1997). Furin is also involved in the processing of bacterial toxins and various cellular and viral proteins. For instance, furin performs proteolysis upon the glycoproteins of HIV, measles, RSV, dengue, and Zika viruses to engender the structural stability of the corresponding proteins, which contributes to the maintenance of viral infectivity (Thomas, 2002; Hoffmann
Consistent with this hypothesis, several HCoVs, such as HCoV-OC43, MERS-CoV, and HCoV-HKU1, all of which cause respiratory infections to varying degrees, contain a furin recognition motif [(R/K)-(2X)n-(R/K)↓] in the S1/S2 cleavage region (Millet and Whittaker, 2014; Le Coupanec
SARS-CoV-2 can successfully proliferate even in cells that express low levels or an absence of the specific protease known to be essential for priming the binding of the S protein to the ACE2, suggesting that an additional protease recognition motif is critical for the enhanced infectivity exhibited by SARS-CoV-2 likely traits of highly pathogenic avian influenza viruses (Munster
Given that furin is expressed in most human tissues, it is conceivable that SARS-CoV-2 infection could occur throughout the body, not confined to the respiratory tract. However, the presence of a furin site in the S protein could not explain the characteristic of SARS-CoV-2 infectivity because less rampant MERS-CoV also has a furin cleavage site in the S protein (Hoffmann
HAT, or TMPRSS11D, is a type 2 transmembrane serine protease known to cleave HA in seasonal and avian influenza viruses (Matsushima
However, it is unclear how HAT regulates the infectivity of SARS-CoV-2. It seems that its function is dispensable and quickly compensated for by other enzymes in that the genetic ablation of HAT did not affect the development of C57BL/6 mice (Bertram
Trypsin is a prototypic serine endopeptidase that cleaves arginine (R) or lysine (K) residues at neutral pH. Although the viral S protein contains these amino acids at numerous sites, trimeric aggregation in the S protein allows only a few trypsin recognition sites to be exposed to the protease. It was reported that trypsin recognizes and cleaves R667 in the S protein of SARS-CoV (Li
Conversely, trypsin treatment before the virus infection decreases SARS-CoV infectivity 10- to 100-fold by cleaving off a part of the S protein (Matsuyama
The most pronounced difference between SARS-CoV and SARS-CoV-2 is their ability to form a syncytium with adjacent cells (Qian
Elastase is expressed as part of the inflammatory response due to viral infection and secreted from neutrophils during severe pneumonia or pseudomonas, resulting in multiple infections within the lungs (Kawabata
Despite increased elastase expression levels, this may not directly affect a structural change of the S protein. Instead,
Matriptase, a type 2 transmembrane serine protease, is highly expressed in epithelial cells within the respiratory tract and the human bronchial epithelial cancer cell line Calu-3. It is well known that matriptase cleaves HA0 of the influenza virus into HA1 and HA2 by recognizing sites of RSSR/RSRR in the MBS motif. Thus, when matriptase expression was inhibited in Calu-3 cells, the infectivity of influenza viruses was considerably decreased (Kawabata
Unlike the proteolytic function of matriptase against HA of influenza viruses that has been well described, the exact role in the cleavage mechanism for various coronaviral S proteins remains explored. Instead, intermittent studies implicate that matriptase may be involved in cleavage at the S1/S2 region in the S protein of MERS-CoV, which is the target of furin and TMPRSS2 (Hamilton
Vimentin is a type 3 intermediate filamentous protein essential for maintaining cytoplasm structure, cell adhesion, cell migration, and cellular signaling (Mor-Vaknin
Vimentin is distributed around the S protein and ACE2 receptor complex. It has been associated with host cell entry by directly interacting with the S and E proteins in SARS-CoV and JEV, respectively (Das
ADAM17, also known as TNF-α-converting enzyme, is a type 1 transmembrane protease whose identified targets are approximately 80 proteins, including cytokines, growth factors, receptors, chemokines, and neurotransmitter regulators (Düsterhöft
ADAM17 has been reported to cleave Arg 708 and Ser 709 of ACE2 proximal to the cell membrane, which leaves intact catalytic activity of ACE2 (Lambert
Given that membrane bound ACE2 plays a receptor function in SARS-CoV-2 infection, it is highly likely that sACE2 affects the pathogenesis of SARS-CoV-2 (Haga
These results suggest that the role of sACE2 produced by ADAM17 might have a limited effect on SARS-CoV-2 infection (Haga
Depending on the types of viruses, diverse mechanisms are involved in regulating virus entry into the host cells. Cellular proteases, including TMPRSS2, HAT, furin, and trypsin, regulate the viral protein binding to the host cell receptors. These enzymes also play an essential role in the SARS-CoV-2 invasion process. However, the functional profile of cellular proteases and the viral infectivity in a particular tissue have not coincided. Many susceptible cell lines to SARS-CoV-2 express none or a low level of specific proteases that are considered critical for the SARS-CoV-2 cell infection (Ou
It has been shown that the glycoproteins of various enveloped viruses, such as coronavirus, HIV, or Newcastle disease virus, enter into cells mediated by endocytosis as well as direct membrane fusion (Fackler and Peterlin, 2000; Cantín
In contrast, HCoV-229E invades fibroblast cells mediated by caveolae, not endocytosis via the clathrin-related-pathway (Nomura, 2005). It employs various methods such as caveolae mediation, flotillin mediation, CLIC/GEECS (CLathrin Independent Carriers/GPI-AP Enriched Endocytic Compartments) mediation micropinocytosis (Gold
Clathrin-mediated or clathrin-independent endocytosis forms an endosome after the virus enters the cell membrane. Cations such as Ca2+ are introduced through a two-pore channel (TPC) of the endosome, and viral S protein is proteolyzed by cathepsin L/B protease at a low-pH. (Sureda
The biological characteristics of SARS-CoV-2 need to be thoroughly investigated to fill the gap between scientific knowledge and effective strategies for the diagnosis, treatment, and prevention of this virus. The advent of the furin cleavage site is attributable to the pathogenesis of SAR-CoV-2 differing from previously known HCoVs. Much more details need to be uncovered to understand which proteases are involved in what stages of infection and how they contribute to virus infection and transmission to other hosts. Although scientists argue that the furin cleavage site in the S protein of the SARS-CoV-2 is necessary only for cell-cell fusion and not for enhancing viral invasion, other proteases, which are associated with an entire life cycle of the virus, are attractive antiviral targets.
The followings need to be considered: 1) although it has been reported that alternative cell entry machinery exists for both clathrin-mediated and clathrin-independent mechanisms, yet another form of endocytosis apart from the two types of machinery has also been identified; 2) various accessory factors are continuously recognized in protease research; 3) distribution of proteases in the tissue and preferred mechanism vary depending on cellular characteristics, tissue specificity, and viral species.
The successful infection of SARS-CoV-2 requires a series of host cell proteases, and thus functional interference of target molecules is an attractive therapeutic strategy for suppressing virus infection. However, as these enzymes account for intrinsic physiologic functions allocated, interrupting their assigned role by therapeutics likely results in an unwanted consequence. Therefore, therapeutics against the host proteases or other proteins should be carefully designed and monitored.
The COVID-19 pandemic caused by SARS-CoV-2 has posed an enormous challenge to public health, and the threat still has a significant impact on humanity. Although prophylactic vaccines are being licensed and commercialized, repeated global epidemics are expected to continue with measurable cases and fatalities. It appears that social distancing in the public sector is still the primary way to reducing viral transmission. As yet, no prominent therapeutics are approved to managing the devastating clinical outcomes associated with this disease. Moreover, the current understanding of the SARS-CoV-2 is limited and has been deduced from the comparison with SARS-CoV, MERS-CoV, and other HCoVs.
Thus, it is clear that further studies are required for the elucidation of viral–host interactions and molecular processes occurring in the cell during and/or after infection to develop an efficient strategy for containment of SARS-CoV-2 infection. Comprehensive progress in understanding biomolecular features linked with clinical practices and immunological characteristics is essential to soothe the current pandemic caused by SARS-CoV-2.
This research was supported by the Korea Disease Control and Prevention Agency Program, Grant No. 2020ER533000, and from the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT, Republic of Korea (Grant No. NRF-2017M3A9E4061995).