SARS-CoV-2 infects human cells through a multi-step process that begins when the virus’s spike protein locks onto a receptor called ACE2 on the cell surface, then relies on host enzymes to cut and activate the spike so it can fuse the viral and cell membranes together. Once fused, the virus dumps its genetic material into the cell and hijacks the cell’s own machinery to make copies of itself. The process is more intricate than that summary suggests, though, involving molecular shape-shifting, multiple backup entry routes, and a set of viral tools that suppress the cell’s alarm systems.
First Contact Is Not with ACE2
Before the spike protein ever touches ACE2, the virus needs to get close to the cell surface and stay there long enough for binding to occur. Recent nanoscopy work shows that this initial docking step depends heavily on sugar-chain molecules called heparan sulfate proteoglycans, which stud the outside of most human cells. In experiments comparing cells with and without ACE2, the amount of virus clinging to the cell surface was essentially the same, and roughly 93 to 94 percent of virus particles landed directly on heparan sulfate clusters. Stripping heparan sulfate from cells with enzymes reduced surface attachment by about 83 to 85 percent, regardless of whether ACE2 was present.1PubMed Central. Nanoscopy reveals heparan sulfate clusters as docking sites for SARS-CoV-2 attachment and entry In other words, heparan sulfate acts like a landing pad that concentrates virus near the cell, giving the spike protein time to find and engage ACE2.
Another molecule that helps the virus get in is neuropilin-1, a receptor found on many cell types including neurons and blood-vessel cells. After the spike protein is cut by the host enzyme furin, one of the fragments carries a short amino-acid sequence that fits neatly into a pocket on neuropilin-1. Depleting neuropilin-1 from cells roughly halved the amount of virus taken up, and tissues rich in neuropilin-1 appear to face higher infection risk.2PubMed Central. The Role of Neuropilin-1 (NRP-1) in SARS-CoV-2 Infection: Review These co-receptors help explain why the virus can infect a surprisingly wide range of tissues beyond the lungs.
The Spike Protein Opens Up
The spike protein is a trimer, meaning three identical copies bundled together, projecting from the virus’s outer envelope. Each copy has a receptor-binding domain (RBD) that can sit in either a “down” position, where it is hidden and unable to contact ACE2, or an “up” position, where it is exposed and ready to bind. The virus essentially flickers between these two states. In the original Wuhan strain, the RBD spends a meaningful fraction of its time in the closed position.3PubMed Central. SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility This flickering is not random wobbling; distant parts of the spike protein influence whether the RBD flips up or stays down, a form of long-range molecular communication within the protein’s structure.4PubMed Central. Distant residues modulate conformational opening in SARS-CoV-2 spike protein
Before the virus even reaches a cell, the spike can also undergo a dramatic, irreversible structural collapse from its “prefusion” form into a “postfusion” form. Cryo-electron microscopy captured both states at near-atomic resolution, showing that the prefusion trimer holds its RBDs clamped down by a segment near the fusion peptide. The transition to the postfusion shape can happen spontaneously, without a target cell, which means some spike molecules burn out before they get a chance to infect anything.5PubMed Central. Distinct conformational states of SARS-CoV-2 spike protein This is one reason why the virus needs many spike proteins on its surface: some will be wasted.
Locking Onto ACE2
When an RBD does flip up and encounters ACE2, the two proteins form an extensive contact surface. The RBD cradles a long helix on the outer face of ACE2, burying a combined surface area of about 1,700 square angstroms, held together by a network of hydrogen bonds and salt bridges. One residue unique to SARS-CoV-2, lysine at position 417, forms a salt bridge with ACE2 that the original SARS virus from 2003 lacked. These subtle contact differences translate into a binding affinity roughly six to seven times tighter for SARS-CoV-2 than for its predecessor.6PubMed Central. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 That tighter grip likely contributed to the pandemic virus’s greater transmissibility.
Variants have pushed binding affinity even further. Molecular dynamics simulations of the Omicron variant’s RBD, which carries numerous mutations at the ACE2 interface, show stronger binding than the original strain, driven by additional hydrogen bonds and a larger buried surface area.7PubMed Central. Mutations on RBD of SARS-CoV-2 Omicron variant result in stronger binding to human ACE2 receptor This matters because ACE2 binding is the gateway to everything that follows.
The Two Cuts That Unlock Fusion
Binding ACE2 is necessary but not sufficient. The spike protein must also be cut by host enzymes at two specific sites before it can fuse with the cell membrane. This “proteolytic priming” is what distinguishes SARS-CoV-2 from many other coronaviruses and is central to its ability to infect human lung cells.
The first cut happens at the junction between the spike’s S1 and S2 subunits, at a site containing a distinctive stretch of basic amino acids. The enzyme furin, which is abundant in human cells, recognizes this multi-basic site and cleaves it. This cut can happen during viral production in an infected cell, so many virus particles arrive at a new cell already pre-cut. The second cut happens at a site called S2′, deeper within the S2 subunit, and this cleavage is strongly enhanced once the spike has already engaged ACE2. A cell-surface enzyme called TMPRSS2 performs this second cut on the outside of the cell.8PubMed Central. Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity In laboratory experiments, knocking down TMPRSS2 expression blocked viral activation in human airway cells, and combining TMPRSS2 inhibitors with furin inhibitors completely prevented infection.9Life Science Alliance. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells
The multi-basic furin site is also essential for the virus to enter human lung cells at all. When researchers mutated it to resemble the simpler cleavage site of the 2003 SARS virus, entry into a TMPRSS2-positive human lung cell line was abolished.10Molecular Cell. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells This furin cleavage site is one of the features that made SARS-CoV-2 so effective at spreading through the human respiratory tract.
Two Doors Into the Cell
Once the spike is primed and ACE2 is engaged, the virus can enter through one of two routes. The primary route in lung tissue involves TMPRSS2 cleaving the spike at S2′ right there on the cell surface, which exposes a “fusion peptide” that punches into the cell membrane and drags the viral envelope toward it until the two membranes merge. This delivers the viral genome directly into the cell’s interior.
The alternative route occurs when the virus is swallowed whole into an endosome, a small bubble that the cell uses to internalize material from its surface. Inside the acidic endosome, a different enzyme called cathepsin L takes over the job of cutting the spike at S2′. Cathepsin L efficiently chops the spike protein in a dose-dependent manner, and the resulting cleavage promotes membrane fusion from within the endosome, releasing the viral RNA into the cell’s cytoplasm.11Signal Transduction and Targeted Therapy. Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development This backup pathway matters because it allows the virus to infect cells that have little TMPRSS2 on their surface.
Which door the virus prefers turns out to depend on the variant. In primary human nasal cultures, all tested variants primarily used the TMPRSS2-dependent surface route, but Omicron was more likely to use the endosomal pathway.12PubMed Central. Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating The Omicron BA.1 and BA.2 sub-lineages favored a TMPRSS2-independent endosomal pathway, and this shift mapped to mutations in the S2 domain of the spike.13PubMed. SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway Delta, by contrast, could exploit yet another entry mechanism involving host metalloproteinases, which Omicron was unable to use.14PubMed Central. Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron These differences in entry route are not just biochemical trivia; they help explain why different variants cause different patterns of disease in the upper versus lower airways.
Hijacking the Cell’s Machinery
Once the viral RNA lands in the cell’s cytoplasm, the cell’s own ribosomes immediately start reading it as if it were a messenger RNA. The first thing translated is an enormous pair of polyproteins, essentially one long chain containing 16 connected protein tools called nonstructural proteins.15PubMed Central. Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness Two viral proteases, known as the main protease and the papain-like protease, then cut this chain at precise locations to release the individual functional proteins.16PubMed Central. SARS-CoV-2 proteases Mpro and PLpro: Design of inhibitors with predicted high potency and low mammalian toxicity using artificial neural networks, ligand-protein docking, molecular dynamics simulations, and ADMET calculations These freed proteins include the RNA-copying enzyme (the RNA-dependent RNA polymerase), a helicase, and several other components needed to build a full replication complex.
The virus then commandeers the cell’s internal membranes. Sections of the endoplasmic reticulum, the cell’s protein-manufacturing network, get reshaped into double-membrane vesicles. These sealed compartments serve as miniature factories where viral RNA replication takes place, shielded from the cell’s antiviral sensors.17PubMed Central. The double-membrane vesicle (DMV): a virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses
A Spell-Checker for RNA
Most RNA viruses mutate rapidly because their copying enzymes are sloppy and lack any error-correction mechanism. Coronaviruses are an exception. SARS-CoV-2 carries a built-in proofreader: an exoribonuclease activity housed within nonstructural protein 14 (nsp14). This enzyme checks the growing RNA strand and clips off incorrectly added nucleotides from the 3′ end, much like a spell-checker that deletes a mistyped letter.18PubMed Central. Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting This proofreading capability is one reason coronaviruses can maintain such large genomes, around 30,000 nucleotides, without collapsing under the weight of accumulated errors. It also has a practical consequence: the proofreader can remove certain antiviral drugs that mimic normal nucleotides after they get incorporated into the growing RNA chain, which is one reason developing effective antivirals against SARS-CoV-2 has been challenging.19PubMed Central. Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN
Escaping the Cell Through an Unusual Exit
After new viral genomes are copied and structural proteins are made, the virus assembles new particles in a compartment between the endoplasmic reticulum and the Golgi apparatus. Getting those new particles out of the cell, however, involves a surprising trick. Most enveloped viruses leave cells through the standard secretory pathway, the same route cells use to ship out proteins. SARS-CoV-2 instead hijacks lysosomes, compartments normally dedicated to digestion and waste disposal, and redirects them to the cell surface for release. This deacidifies the lysosomes and disables the degradation enzymes inside them, which disrupts the cell’s ability to process and present viral fragments to the immune system.20Cell. β-Coronaviruses Traffic to Lysosomes for Egress
A specific viral protein called ORF3a actively promotes this lysosomal exit strategy. Interestingly, this ability appears to be unique to SARS-CoV-2’s version of ORF3a; the equivalent protein from the 2003 SARS virus does not promote lysosomal exocytosis in the same way.21PubMed Central. ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress This unconventional exit pathway may have contributed to the virus’s success by simultaneously releasing new viruses and weakening the infected cell’s immune signaling.
Spreading Without Leaving the Cell
SARS-CoV-2 does not rely solely on releasing free-floating virus particles to spread. Infected cells that display spike protein on their surface can fuse directly with neighboring ACE2-positive cells, forming large multinucleated masses called syncytia. Expression of the spike protein alone, without any other viral protein, is sufficient to trigger this fusion.22PubMed Central. Syncytia formation by SARS-CoV-2-infected cells This cell-to-cell transmission route is more efficient for SARS-CoV-2 than it was for the 2003 SARS virus, partly because the SARS-CoV-2 spike is better at driving cell-cell fusion.23PubMed Central. SARS-CoV-2 spreads through cell-to-cell transmission Cell-to-cell spread can help the virus dodge antibodies circulating in the fluid between cells, since the virus never has to travel through that exposed space.
Silencing the Cell’s Alarm System
A healthy cell detects viral RNA inside it and responds by producing interferons, signaling molecules that warn neighboring cells and activate antiviral defenses. SARS-CoV-2 aggressively suppresses this response. Studies found that interferon and interferon-stimulated gene expression were poorly induced during infection, and once the virus was established, cells became highly resistant to interferon even when it was added artificially from outside.24PubMed Central. SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors The nonstructural protein nsp1 plays a central role: it blocks interferon production partly by preventing a key signaling molecule (IRF3) from being activated, and it also depletes proteins that relay the interferon signal inside the cell. In a screen of all 26 SARS-CoV-2 proteins, nsp1 and the accessory protein ORF6 were the most potent interferon blockers, suppressing interferon signaling by about 98 and 91 percent respectively.25Cell Reports. Evasion of Type I Interferon by SARS-CoV-2 This suppression buys the virus a critical window of time to replicate before the immune system fully activates.
The Glycan Shield
The spike protein does not present a naked protein surface to the immune system. Each spike subunit carries 22 sites where sugar chains (glycans) are chemically attached, and these sites have remained highly conserved as the virus has evolved.26PubMed Central. Disruption of spike protein N-glycosylation induces its endoplasmic reticulum retention and attenuates SARS-CoV-2 infectivity Mass spectrometry mapping of these glycans revealed a complex landscape of sugar structures across the trimer.27PubMed Central. Site-specific glycan analysis of the SARS-CoV-2 spike These sugars serve a dual purpose. They help the spike protein fold correctly during production, and they physically mask protein surfaces that antibodies would otherwise target. Removing the glycan at one particular site, N343, made the virus about five times more sensitive to neutralizing antibodies from people who had recovered from COVID. Vaccination after infection, however, generated antibodies that could overcome the glycan’s protective effect.28PubMed Central. SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity The glycan shield is essentially camouflage: the virus coats itself in the same kinds of sugar molecules that human cells use, making it harder for the immune system to distinguish friend from foe.
Where in the Body the Virus Can Strike
The virus’s dependence on ACE2 and TMPRSS2 means infection is concentrated in tissues where both are present. You might expect the lungs to top the list, but ACE2 expression in the respiratory system is actually limited, observed at low levels in only a subset of cells in some individuals. ACE2 is much more abundant in the small intestine’s absorptive cells, kidney tubules, the gallbladder, heart muscle cells, and male reproductive cells.29PubMed Central. The protein expression profile of ACE2 in human tissues The two receptors also do not perfectly overlap: ACE2 and TMPRSS2 co-localize in the gastrointestinal tract, kidneys, reproductive organs, and a specific type of lung cell called AT2 cells. Adding another layer of complexity, ACE2 production is boosted by estrogen while TMPRSS2 depends on androgens.30Biochemical Society Transactions. Effects and regulation of ACE2 and TMPRSS2 abundance in healthy humans and in patients with SARS-CoV-2 This hormonal regulation may partially explain some of the observed sex differences in COVID-19 severity.
How Drugs Target These Steps
Understanding each stage of the infection cycle has given researchers specific molecular targets to aim at. The two viral proteases that chop the polyprotein chain into functional pieces are prime drug targets because, without them, the virus cannot assemble its replication machinery. Nirmatrelvir, the active ingredient in Paxlovid, works by blocking the main protease, preventing it from processing the polyproteins needed for replication. It is packaged with ritonavir, which slows the body’s breakdown of nirmatrelvir so it stays active longer.31Biomedicine & Pharmacotherapy. Review Paxlovid (Nirmatrelvir/Ritonavir): A new approach to Covid-19 therapy? The papain-like protease is also a promising target, though inhibitors against it have lagged behind in development and, until recently, had not demonstrated effectiveness in animal models.32PubMed Central. Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model
Other antivirals target the RNA-copying enzyme directly. Remdesivir mimics a building block of RNA and gets incorporated into the growing strand, disrupting further copying. Molnupiravir and favipiravir work by a related strategy, also targeting the RNA polymerase but introducing errors into the copied genome rather than simply stalling the enzyme.33PubMed Central. Carboxylesterase Factors Influencing the Therapeutic Activity of Common Antiviral Medications Used for SARS-CoV-2 Infection The virus’s proofreading exonuclease can partially counteract drugs like remdesivir by excising the fake nucleotide, which is one reason these drugs must reach high enough concentrations to overwhelm the proofreader.
Why ACE2 Shape Determines Which Species Get Infected
The specifics of the spike-ACE2 handshake also dictate which animal species are vulnerable. Researchers have modeled ACE2 proteins from dozens of vertebrates to identify which ones fit the spike’s binding surface. Small differences in just a few amino acids at the contact points can dramatically change susceptibility. White-tailed deer ACE2, for example, binds well enough that deer populations across North America have sustained widespread SARS-CoV-2 transmission. Elk ACE2, despite being closely related, carries a single substitution at a key position that weakens spike binding.34PubMed Central. Species- and variant-specific ACE2 compatibility shapes SARS-CoV-2 spillover potential in North American cervids But receptor compatibility is only one piece of the puzzle. A species also needs the right proteases in the right tissues, a mode of contact that allows virus transmission, and an immune system that fails to contain the virus rapidly. Marine mammals, for instance, may have compatible ACE2 at the molecular level yet rarely encounter the virus in their environment.35PubMed Central. Systematic multi-reference vertebrate ACE2 sequence similarity analysis predicts species susceptibility to SARS-related sarbecoviruses Predicting the next animal reservoir therefore requires looking well beyond the receptor.