SARS-CoV-2, human immunodeficiency virus, influenza virus, hepatitis C virus, and hepatitis B virus differ in genome composition, entry pathways, and replication strategies. Interventions targeting receptor binding, membrane fusion, nucleic acid replication, protein processing, genome integration, capsid assembly, and viral release represent major directions in antiviral drug development and mechanistic research.

Keywords: SARS-CoV-2; HIV; influenza virus; HCV; HBV; viral replication; viral protease; RNA polymerase; reverse transcriptase; antiviral drug

1 Viral Structure, Replication Modes, and Antiviral Targets

1.1 Basic Viral Structure

Viral particles generally consist of a DNA or RNA genome enclosed within a protein capsid. Some viruses also possess a lipid envelope derived from the host-cell membrane. Viral glycoproteins on the envelope recognize host receptors and mediate viral entry, whereas the capsid protects the viral genome and participates in uncoating, intracellular transport, viral assembly, and maturation. Viruses lack independent energy metabolism, protein-translation machinery, and complete replication systems and must enter host cells and use host ribosomes, membrane structures, nucleotides, and energy resources to replicate.

1.2 Viral Genomes and Replication Modes

Viral genomes can be divided into DNA and RNA genomes and further classified as single-stranded, double-stranded, positive-sense, negative-sense, or segmented genomes. Positive-sense RNA can directly function as messenger RNA for protein translation, whereas negative-sense RNA must first be converted into complementary positive-sense RNA by a viral RNA-dependent RNA polymerase. Retroviruses first reverse-transcribe RNA into DNA and then integrate viral DNA into the host chromosome. Although HBV is a DNA virus, its replication requires transcription of a pregenomic RNA intermediate, which is subsequently reverse-transcribed by the viral polymerase to generate new viral DNA.

1.3 Major Stages Targeted by Antiviral Drugs

(1) Viral Attachment and Entry

Viruses first recognize host-cell receptors and coreceptors through envelope glycoproteins or capsid proteins. Blocking interactions between viral ligands and host receptors, inhibiting host-protease activation of viral glycoproteins, or interfering with fusion between the viral and cellular membranes can reduce viral entry.

(2) Viral Nucleic Acid Replication

RNA viruses generally depend on viral RNA-dependent RNA polymerases, whereas retroviruses and HBV depend on reverse transcriptase or viral polymerases with reverse-transcription activity. Nucleoside or nucleotide analogues can competitively enter viral nucleic acid synthesis and cause chain-extension arrest, delayed termination, or accumulation of mutational errors.

(3) Viral Protein Processing

Some RNA viruses and retroviruses initially translate large polyproteins, which are subsequently cleaved by viral proteases into mature functional proteins. Inhibition of viral proteases prevents maturation of replication enzymes, structural proteins, and accessory proteins, thereby interrupting viral replication.

(4) Assembly, Maturation, and Release

Viral nucleic acids and structural proteins must assemble into nucleocapsids in a defined sequence and undergo envelope acquisition, proteolytic maturation, or budding from host membranes to form infectious particles. Capsid modulators, maturation inhibitors, and viral-release inhibitors can act at this stage.

2 Severe Acute Respiratory Syndrome Coronavirus 2

2.1 Viral Structure and Genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the genus Betacoronavirus within the family Coronaviridae and is an enveloped positive-sense single-stranded RNA virus. Its major structural proteins include spike protein S, membrane protein M, envelope protein E, and nucleocapsid protein N. Nonstructural proteins include the 3C-like protease (3CLpro), also known as the main protease (Mpro), the papain-like protease (PLpro), helicase, and RNA-dependent RNA polymerase (RdRp). The S protein mediates receptor recognition and membrane fusion, the N protein binds genomic RNA, and the M and E proteins participate in viral-particle assembly and budding.

Figure 1 Schematic representation of SARS-CoV-2 virion structure

2.2 Viral Entry and Replication

The receptor-binding domain of the S protein first recognizes angiotensin-converting enzyme 2 on the host-cell surface. The S protein is then cleaved by host proteases and undergoes conformational changes that promote fusion of the viral envelope with the plasma membrane or endosomal membrane. After viral RNA is released into the cytoplasm, it is translated directly to generate replicase polyproteins. The 3CLpro and PLpro proteases cleave these polyproteins into mature nonstructural proteins, which assemble into the replication-transcription complex. RdRp synthesizes new genomic and subgenomic RNAs. The membrane structural proteins S, M, and E are synthesized on ER-associated ribosomes and enter the ER–Golgi intermediate compartment, whereas the N protein is synthesized in the cytosol and forms ribonucleoprotein complexes with genomic RNA. Newly formed virions assemble mainly at the ER–Golgi intermediate compartment and are subsequently released through intracellular vesicular transport.

2.3 Major Drug Targets

(1) S Protein and Viral Entry

Intervention against the receptor-binding region of the S protein, the interaction interface with host ACE2, or conformational changes involved in membrane fusion can reduce viral attachment to host cells. Such strategies include receptor-binding blockers, fusion-inhibitory peptides, and S protein-targeting antibodies.

(2) 3CLpro and PLpro

The 3CLpro and PLpro proteases cleave viral replicase polyproteins and are essential for coronavirus replication. The relatively well-defined substrate-recognition site of 3CLpro makes it suitable for structure-based inhibitor screening. In addition to processing viral polyproteins, PLpro may affect host ubiquitination and interferon-related signaling.

(3) RNA-Dependent RNA Polymerase

RdRp is the core enzyme responsible for viral RNA replication. After entering cells, nucleoside or nucleotide analogues are converted into active triphosphate forms, compete with natural nucleotides, and cause extension arrest or delayed termination during viral RNA synthesis.

(4) Drug Repurposing and In Vitro Screening

Lopinavir, ritonavir, saquinavir, nelfinavir, carfilzomib, and other compounds have been used in virtual screening, molecular docking, or drug-repurposing studies targeting coronavirus proteases. Computational studies identified nelfinavir as a potential inhibitor of the SARS-CoV-2 main protease, but predicted binding affinity cannot replace enzymatic, cell-infection, and in vivo efficacy validation. Remdesivir and chloroquine diphosphate showed in vitro inhibitory activity in early cell-infection systems. Remdesivir mainly targets viral RdRp, whereas chloroquine diphosphate may affect endosomal and lysosomal environments as well as multiple host-cell processes.

2.4 SARS-CoV-2-Related Products

3 Human Immunodeficiency Virus

3.1 Viral Structure and Cell Entry

Human immunodeficiency virus (HIV) is a retrovirus whose particles contain two copies of a positive-sense single-stranded RNA genome. Its envelope glycoprotein complex consists of gp120 and gp41. Gp120 first binds CD4 on the host-cell surface and subsequently interacts with the CCR5 or CXCR4 coreceptor. Receptor binding induces conformational changes in the envelope glycoprotein, allowing the gp41 fusion peptide to insert into the host-cell membrane. Gp41 then folds into a six-helix bundle, bringing the viral envelope and cell membrane into proximity and completing membrane fusion.

3.2 Reverse Transcription and Genome Integration

After the viral core enters the cytoplasm, uncoating occurs. Reverse transcriptase uses viral RNA as a template to synthesize complementary DNA and subsequently generates double-stranded viral DNA. Viral DNA forms a preintegration complex with integrase and other viral and host proteins and is transported into the nucleus. Integrase catalyzes viral-DNA end processing and strand transfer, inserting viral DNA into the host chromosome to form a long-lived provirus. The provirus uses the host transcriptional machinery to produce new viral RNAs, structural proteins, and viral enzymes.

3.3 Viral Assembly and Maturation

HIV genomic RNA, Gag polyprotein, Gag-Pol polyprotein, and envelope proteins assemble near the plasma membrane, and immature viral particles leave the cell by budding. Viral protease subsequently cleaves the Gag and Gag-Pol polyproteins, allowing matrix protein, capsid protein, nucleocapsid protein, reverse transcriptase, integrase, and protease to rearrange and form infectious mature particles. HIV can be targeted at multiple stages, including attachment, coreceptor binding, membrane fusion, reverse transcription, integration, proteolytic processing, and maturation. Consequently, related drugs are generally used in combinations that act on different targets.

3.4 HIV-Related Products

4 Influenza Virus

4.1 Viral Structure and Classification

Influenza viruses belong to the family Orthomyxoviridae and are enveloped viruses with segmented negative-sense RNA genomes. Their envelopes mainly contain hemagglutinin (HA) and neuraminidase (NA), and influenza A virus also contains the M2 proton channel. Internal components include nucleoprotein NP, matrix protein M1, and an RNA polymerase complex composed of PB1, PB2, and PA. Influenza viruses are divided into types A, B, C, and D according to the antigenicity of their nucleoprotein and matrix protein. Influenza A viruses are further classified into subtypes according to HA and NA antigen types.

Figure 2 Schematic representation of influenza virus structure and genome organization

4.2 Viral Entry, Replication, and Release

HA recognizes sialic acid receptors on the host-cell surface and mediates viral endocytosis. Endosomal acidification induces conformational changes in HA and promotes fusion of the viral envelope with the endosomal membrane. The M2 proton channel promotes acidification within the viral particle, allowing viral ribonucleoprotein complexes to dissociate from M1 and be released into the cytoplasm. Viral ribonucleoprotein complexes then enter the nucleus, where transcription and genome replication are mediated by the viral polymerase. Newly synthesized viral RNA, nucleoprotein, polymerase, and envelope proteins assemble near the plasma membrane. NA cleaves sialic acid residues from the cell surface, promoting release of progeny virions from host cells.

4.3 Major Drug Targets

(1) M2 Proton Channel

Amantadine and rimantadine bind the M2 proton channel of influenza A virus and inhibit internal acidification and viral uncoating. Amino acid substitutions in M2 can markedly reduce drug binding and confer resistance.

(2) Neuraminidase

Zanamivir, oseltamivir, and laninamivir occupy the NA catalytic site and inhibit cleavage of sialic acid residues, preventing newly formed virions from being released efficiently from the cell surface and reducing spread to neighboring cells.

(3) Viral RNA Polymerase

After entering cells, favipiravir is converted into an active nucleoside triphosphate form that is recognized and incorporated by the viral RNA polymerase. This can inhibit RNA-chain elongation or increase the mutational burden of the viral genome.

(4) Membrane Fusion and Host Signaling

Arbidol can affect viral-envelope protein conformation and the membrane-fusion process. Influenza infection also activates MAPK, NF-κB, PI3K/AKT, and interferon-related signaling pathways. These host pathways regulate viral gene expression, inflammation, cell survival, and viral release and are therefore important directions in host-targeted antiviral research.

4.4 Influenza Virus-Related Products

5 Hepatitis C Virus

5.1 Viral Structure and Cell Entry

Hepatitis C virus (HCV) belongs to the family Flaviviridae and is an enveloped positive-sense single-stranded RNA virus that primarily infects hepatocytes. Viral particles sequentially interact with host factors including scavenger receptor class B type I, CD81, Claudin-1, and Occludin and enter cells through clathrin-mediated endocytosis. Endosomal acidification promotes fusion of the viral envelope with the endosomal membrane, and uncoating releases genomic RNA into the cytoplasm. Because HCV RNA itself functions as messenger RNA, it can directly initiate viral-protein translation.

5.2 Polyprotein Processing and RNA Replication

The HCV genome encodes a large polyprotein that is subsequently cleaved by host proteases and viral NS2 and NS3/4A proteases to generate mature core protein, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS3/4A processes the viral polyprotein and can also interfere with host innate immune signaling. NS4B induces formation of membranous replication platforms, NS5A participates in replication-complex organization, RNA replication, and viral-particle formation, and NS5B functions as the RNA-dependent RNA polymerase that catalyzes viral RNA synthesis.

5.3 Viral Assembly and Direct-Acting Targets

Newly synthesized HCV RNA and core protein undergo initial assembly near lipid droplets and coordinate with host very-low-density lipoprotein synthesis and secretion to form infectious lipoviral particles. HCV direct-acting antiviral compounds are mainly classified as NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors. Host factors such as Cyclophilin A can also serve as indirect intervention targets. Combining compounds from multiple classes simultaneously suppresses viral protein processing, replication-complex formation, and RNA-chain elongation and reduces the effect of individual resistance mutations.

5.4 HCV-Related Products

6 Hepatitis B Virus

6.1 Viral Genome and cccDNA

Hepatitis B virus (HBV) belongs to the family Hepadnaviridae and is an enveloped virus with a partially double-stranded relaxed circular DNA genome. After the virus enters hepatocytes, the nucleocapsid transports relaxed circular DNA into the nucleus. Host repair enzymes remove the viral polymerase and RNA primer, fill the DNA gaps, and generate covalently closed circular DNA (cccDNA). cccDNA persists as a minichromosome and serves as the principal template for continuous viral-RNA transcription. Its long-term stability is a major molecular basis for persistent HBV infection and viral rebound after treatment discontinuation.

Figure 3 Schematic representation of HBV genome organization and functional regions

6.2 Pregenomic RNA and Reverse Transcription

cccDNA is transcribed into pregenomic RNA, surface-antigen RNAs, and other viral transcripts. Pregenomic RNA functions both as a translation template for core protein and polymerase and as a genome precursor packaged together with viral polymerase into newly forming nucleocapsids. Within the nucleocapsid, the viral polymerase first synthesizes negative-strand DNA using pregenomic RNA as the template while degrading the RNA template. It then synthesizes an incomplete positive-strand DNA, ultimately generating a new relaxed circular DNA genome.

6.3 Viral Particles and Antigen Release

Nucleocapsids containing mature viral DNA acquire envelopes composed of large, middle, and small surface proteins and are released as infectious Dane particles. Some nucleocapsids return to the nucleus and replenish the cccDNA pool. Infected cells also release large quantities of spherical or filamentous subviral particles lacking complete nucleocapsids. These particles mainly consist of HBsAg and are the principal source of the abundant circulating surface antigen detected in infected individuals.

6.4 Major Drug Targets

(1) HBV Polymerase

Nucleotide analogues such as Tenofovir and Adefovir are converted into active forms within cells, compete with natural substrates, and are incorporated by the viral polymerase into newly synthesized DNA, thereby inhibiting reverse transcription and viral-DNA synthesis.

(2) Core Protein and Capsid Assembly

Core protein allosteric modulators bind the core-protein dimer interface or capsid allosteric pockets, alter capsid-assembly kinetics, induce formation of aberrant capsids, or prevent proper packaging of pregenomic RNA and polymerase.

(3) Viral RNA and HBsAg Expression

Interference with HBV RNA tailing, stability, and degradation can reduce viral-transcript levels and subsequently decrease expression of HBsAg and other viral proteins. Research compounds such as RG7834 are mainly used to investigate PAPD5/PAPD7-related RNA-stability mechanisms.

(4) Host Immune Receptors

TLR7 and TLR8 agonists can stimulate dendritic cells, monocytes, and other immune cells to produce cytokines and enhance innate and subsequent adaptive immune responses. Their antiviral effects must be evaluated together with the intensity of immune activation, hepatic inflammation, and the safety range.

6.5 HBV-Related Products

7 Comparison of Targeted Strategies for the Five Viruses

The five viruses differ substantially in receptor recognition, genome replication, and viral-particle maturation. Major intervention stages for SARS-CoV-2 include S protein-mediated entry, viral-protease processing, and RdRp-mediated RNA replication. HIV can be inhibited at multiple stages, including attachment, membrane fusion, reverse transcription, integration, and maturation. Influenza antiviral drugs mainly target the M2 proton channel, neuraminidase, viral polymerase, and membrane-fusion process. HCV can be blocked at multiple steps through direct-acting targets including NS3/4A, NS5A, and NS5B. HBV treatment research must simultaneously address reverse transcription, capsid assembly, viral-RNA stability, and the persistence of cccDNA.

References

[1] Xu Z, et al. Nelfinavir was predicted to be a potential inhibitor of 2019-nCoV main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation. bioRxiv.

[2] Wang M, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30:269-271.

[3] Wilen CB, Tilton JC, Doms RW. HIV: Cell Binding and Entry. Cold Spring Harb Perspect Med. 2012;2(8):a006866.

[4] Hutchinson EC. Influenza Virus. Trends Microbiol. 2018;26(9):809-810.

[5] Ludwig S, et al. Influenza-Virus-Induced Signaling Cascades: Targets for Antiviral Therapy? Trends Mol Med. 2003;9(2):46-52.

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