PLpro in SARS-CoV-2: Structure, Function, and Immune Impact

SARS-CoV-2, the virus responsible for COVID-19, relies on key enzymes to replicate and evade host defenses. One of these is papain-like protease (PLpro), a multifunctional protein essential for viral processing and immune system interference. Understanding its structure and function provides insights into viral mechanisms and potential therapeutic targets.

Beyond its enzymatic role, PLpro modulates the host immune response, making it a promising drug target.

Structural Organization

PLpro, a cysteine protease encoded within the SARS-CoV-2 genome, is a domain of nonstructural protein 3 (nsp3), a key component of the viral replication-transcription complex. It adopts a conserved papain-like fold with a right-handed thumb-palm-fingers architecture. A zinc-binding motif within the fingers subdomain stabilizes its structure and catalytic function. High-resolution crystallographic studies have shown that four conserved cysteine residues coordinate zinc, ensuring structural rigidity.

The catalytic core resides in the palm subdomain, where a triad of cysteine, histidine, and aspartate facilitates peptide bond cleavage. This active site, housed in a deep groove, allows precise substrate recognition. Structural comparisons with PLpro homologs from SARS-CoV and MERS-CoV show high conservation in this region, though variations in loop flexibility and surface charge distribution influence substrate specificity. Cryo-electron microscopy and X-ray crystallography have revealed subtle differences in the substrate-binding pocket that enhance interaction with viral polyproteins.

PLpro also contains an N-terminal ubiquitin-like (Ubl) domain, which may regulate protein interactions. Structurally similar to host ubiquitin, this domain is connected to the catalytic core via a flexible linker, allowing dynamic conformational changes. Nuclear magnetic resonance (NMR) spectroscopy has shown that this domain shifts upon substrate binding, suggesting a potential allosteric regulatory mechanism.

Active Site Features

The active site of SARS-CoV-2 PLpro is a well-defined pocket that governs substrate recognition and catalysis. The catalytic triad—cysteine (Cys111), histidine (His272), and aspartate (Asp286)—orchestrates peptide bond cleavage through a nucleophilic attack mechanism. Structural analyses reveal that the active site groove is highly conserved among coronaviruses, though subtle differences influence substrate binding and enzymatic efficiency. The histidine stabilizes the thiolate form of cysteine, enhancing reactivity, while the aspartate ensures proper proton transfer.

A substrate-binding cleft, defined by loops and secondary structures, dictates substrate selectivity. Comparisons between SARS-CoV and SARS-CoV-2 PLpro show a shift in the loop adjacent to the S3-S4 subsites, altering enzyme preference for certain peptide sequences. This affects cleavage efficiency and replication kinetics. High-resolution studies have identified key hydrogen bonds and van der Waals forces that stabilize substrate binding, underscoring the precision of molecular recognition.

The active site also includes an oxyanion hole, which stabilizes the tetrahedral transition state during catalysis. Formed by backbone amides, this region creates an electrostatic environment favorable for intermediate stabilization. Mutagenesis studies indicate that alterations in this region significantly reduce catalytic efficiency. Conserved water molecules near the active site may participate in proton shuttling, fine-tuning the reaction mechanism.

Role in Polyprotein Processing

SARS-CoV-2 relies on proteolytic cleavage to generate functional viral proteins, with PLpro playing a key role. The virus encodes its replicative machinery as two large polyproteins, pp1a and pp1ab, which require precise cleavage to yield essential nonstructural proteins. PLpro processes three specific sites, releasing nsp1, nsp2, and nsp3. These cleavage events are highly conserved among betacoronaviruses, ensuring accurate viral maturation.

Comparative enzymatic assays show that SARS-CoV-2 PLpro has slightly higher catalytic efficiency at certain cleavage sites than its SARS-CoV counterpart, potentially influencing replication rates. The protease’s substrate preference is dictated by its active site architecture, particularly the S1 and S2 subsites. Mutagenesis studies confirm that minor alterations in these regions significantly reduce cleavage efficiency.

Beyond enzymatic function, PLpro-mediated cleavage is crucial for assembling the replication-transcription complex. The timely release of nsp1, nsp2, and nsp3 supports the formation of double-membrane vesicles where viral RNA synthesis occurs. Structural and biochemical studies highlight how conformational dynamics influence its catalytic cycle, with substrate binding triggering subtle rearrangements that optimize cleavage efficiency.

Links to Host Immune Modulation

PLpro also manipulates host immune pathways to create a favorable environment for viral replication. It cleaves ubiquitin and interferon-stimulated gene 15 (ISG15) conjugates from host proteins, disrupting key immune signaling pathways. By removing ubiquitin from proteins like TRAF3 and TRAF6, PLpro inhibits NF-κB and type I interferon responses, dampening antiviral defenses.

Its deISGylation activity further suppresses immunity by interfering with ISG15-mediated stabilization of antiviral proteins, including those in JAK-STAT signaling. Structural comparisons indicate that SARS-CoV-2 PLpro has enhanced deISGylation efficiency compared to SARS-CoV, which may contribute to its immune evasion capabilities.

Variation Among Coronaviruses

PLpro differs across coronaviruses, reflecting evolutionary adaptations affecting replication and immune interactions. While the overall fold and catalytic mechanism are conserved, sequence variations in key structural elements alter substrate specificity and enzymatic efficiency. SARS-CoV-2 PLpro shares approximately 83% sequence identity with SARS-CoV PLpro, yet differences in surface charge and loop dynamics impact biochemical properties.

Notably, the S3-S4 subsites in SARS-CoV-2 PLpro exhibit minor shifts that enhance affinity for viral polyprotein cleavage sites. These differences may contribute to variations in replication efficiency and pathogenicity.

MERS-CoV PLpro, in contrast, shows greater structural divergence, particularly in substrate recognition regions. It has a reduced ability to cleave ISG15, suggesting a distinct immune evasion strategy. Variations in zinc-binding domain stability further influence protease activity and structural rigidity. These evolutionary differences highlight the need for species-specific therapeutic strategies targeting PLpro.

Comparison With 3CLpro

SARS-CoV-2 encodes two major proteases, PLpro and 3CLpro, each with distinct roles in viral processing. PLpro cleaves the nsp1/nsp2, nsp2/nsp3, and nsp3/nsp4 junctions, while 3CLpro processes the remaining nonstructural proteins. Structurally, 3CLpro adopts a chymotrypsin-like fold, differing from the papain-like architecture of PLpro. It also utilizes a cysteine-histidine dyad instead of PLpro’s cysteine-histidine-aspartate triad, contributing to distinct substrate specificities.

Unlike PLpro, 3CLpro has minimal interaction with host immune pathways. While PLpro deubiquitinates and deISGylates host proteins to suppress immune signaling, 3CLpro primarily functions as a dedicated viral protease. This distinction is important for drug development—3CLpro inhibitors can focus solely on disrupting viral replication, while PLpro inhibitors must consider host-pathogen interactions.

Structural and biochemical studies indicate that 3CLpro is a more conserved therapeutic target, with broad-spectrum inhibitors showing efficacy against multiple coronaviruses. In contrast, PLpro-targeting drugs require a nuanced approach due to its role in immune modulation.