Viral replication

Viral replication is the process by which viruses, as obligate intracellular parasites, hijack the machinery of host cells to produce progeny virions capable of infecting new cells and propagating the infection.^[1] This cycle is essential for viral survival and spread, involving a series of coordinated stages that exploit host resources while minimizing detection by the immune system.^[2] The replication process begins with attachment, where the viral particle (virion) binds to specific receptors on the host cell surface via viral glycoproteins or capsid proteins, determining host tropism and tissue specificity.^[1] Following attachment, penetration occurs, either through direct fusion of the viral envelope with the host membrane (common in enveloped viruses like HIV) or via endocytosis, where the virion is engulfed into an endosome.^[2] Once inside, uncoating releases the viral genome—either DNA or RNA—from its protective capsid, allowing access to the cellular environment.^[1] Subsequent stages focus on genome replication and gene expression, which vary significantly by viral type according to the Baltimore classification system, encompassing seven groups based on nucleic acid type and replication strategy (e.g., double-stranded DNA viruses like herpesviruses replicate in the nucleus using host polymerases, while positive-sense single-stranded RNA viruses like coronaviruses use their own RNA-dependent RNA polymerase in the cytoplasm).^[3] Viral proteins, including structural components and enzymes, are synthesized using host ribosomes and translation machinery,^[1] often leading to the shutdown of host protein production to prioritize viral needs.^[4] Assembly then packages the replicated genomes into new capsids, sometimes with envelopes acquired from host membranes, forming mature virions.^[2] Finally, release disperses the progeny through cell lysis (non-enveloped viruses) or budding (enveloped viruses), which preserves the envelope but may attenuate infectivity.^[1] Key variations in replication highlight viral diversity: DNA viruses generally integrate or replicate episomally in the nucleus, while most RNA viruses operate in the cytoplasm to avoid host defenses, though exceptions like retroviruses reverse-transcribe RNA to DNA for nuclear integration.^[3] Replication fidelity differs markedly, with RNA viruses exhibiting high mutation rates due to error-prone polymerases, fostering rapid evolution and immune evasion, whereas DNA viruses achieve greater accuracy.^[3] These processes not only drive pathogenesis but also underpin antiviral strategies, such as targeting entry receptors or polymerases.^[2]

Overview

Definition and scope

Viral replication is the process by which viruses produce progeny virions, utilizing the host cell's biosynthetic machinery to replicate their genetic material and assemble new infectious particles.^[1] This obligatory intracellular parasitism distinguishes viruses from autonomous replicating organisms, as they lack the ribosomes and enzymes necessary for independent propagation.^[5] A prerequisite for viral replication is the structure of the virion, the complete extracellular infectious form of the virus, which consists of a nucleic acid genome (either DNA or RNA) enclosed by a protein capsid that provides protection and facilitates delivery to host cells; some virions also feature a surrounding lipid envelope acquired from the host membrane.^[5] The genome encodes viral proteins essential for replication, including those for capsid formation and, in enveloped viruses, glycoproteins embedded in the lipid layer.^[5] The scope of viral replication encompasses various modes, such as the lytic cycle—in which the viral genome directs rapid production and release of virions through host cell lysis—and, in bacteriophages, the lysogenic cycle, where the viral genome integrates into the host's genetic material (as a prophage) and replicates passively alongside it without immediate cell destruction; viruses infecting eukaryotic cells often exhibit analogous productive and latent phases.^[6] This framework applies specifically to viruses as acellular entities with nucleic acid genomes and does not extend to bacterial conjugation, a direct cell-to-cell transfer of plasmid DNA enabling horizontal gene exchange among bacteria, or prion propagation, which relies on conformational changes in host proteins without nucleic acid involvement.^[7][8] The foundational understanding of viral replication emerged in early 20th-century virology, particularly through Félix d'Herelle's 1917 discovery of bacteriophages—filterable agents that lysed bacterial cultures—demonstrating for the first time a virus-like mechanism of propagation using bacterial hosts as machinery.^[9] D'Herelle's observations of serial dilution experiments revealing self-propagating lytic activity coined early concepts akin to modern viral replication, shifting virology from mere pathogen identification to mechanistic inquiry.^[9]

Biological significance

Viral replication plays a central role in pathogenesis by directly damaging host cells and facilitating disease progression. In many cases, the production of new viral particles culminates in cell lysis, where the host cell membrane ruptures to release progeny viruses, leading to tissue destruction and symptoms such as inflammation and organ failure seen in infections like influenza or poliovirus.^[10] Viruses also employ immune evasion strategies during replication, such as downregulating major histocompatibility complex (MHC) class I molecules to avoid detection by cytotoxic T cells or inducing latency to persist without active replication, as observed in herpesviruses.^[11] Furthermore, certain viruses contribute to oncogenesis by integrating their genomes into host DNA or expressing oncoproteins that disrupt cell cycle control; for instance, high-risk human papillomavirus (HPV) types replicate in epithelial cells and produce E6 and E7 proteins that inactivate tumor suppressors p53 and Rb, driving cervical cancer development in over 90% of cases.^[12] Beyond disease causation, viral replication has profoundly shaped evolution through horizontal gene transfer (HGT), where viral particles shuttle genetic material between organisms, introducing novel genes that enhance host adaptability or virulence. This process has been a key driver of genetic diversity, with evidence from bacteriophages transferring antibiotic resistance genes among bacteria and eukaryotic viruses exchanging sequences that influence immune responses or metabolic pathways in hosts.^[13] For example, endogenous retroviral elements, remnants of ancient infections, comprise up to 8% of the human genome and have contributed functional genes involved in placental development and innate immunity.^[14] Viral replication principles underpin therapeutic applications, particularly in gene therapy and vaccine development. Adeno-associated virus (AAV) vectors, derived from non-pathogenic parvoviruses, exploit controlled replication machinery to deliver therapeutic genes into target cells without integrating into the host genome, enabling long-term expression for treating conditions like spinal muscular atrophy, as approved in therapies like Zolgensma.^[15] Similarly, self-amplifying mRNA (saRNA) vaccines incorporate replicase genes from alphaviruses to mimic viral RNA replication within cells, amplifying antigen production for robust immune responses; these have shown promise in preclinical models for influenza and COVID-19, requiring lower doses than conventional mRNA vaccines.^[16] The global health and economic ramifications of unchecked viral replication are exemplified by the COVID-19 pandemic, caused by SARS-CoV-2, which from 2020 to 2025 resulted in over 7 million confirmed deaths and substantial economic losses worldwide due to healthcare costs, lost productivity, and supply chain disruptions.^[17] The emergence of variants like Delta (2021) and Omicron (2021-2022), driven by replication errors in immune hosts, increased transmissibility and immune escape, prolonging the crisis and necessitating updated vaccines; by 2025, ongoing surveillance tracks subvariants such as XFG and KP.3.1.1, with the pandemic contributing to a 25% rise in global anxiety and depression prevalence linked to its stressors.^[18][19]

General replication cycle

Attachment and entry

Viral attachment is the initial step in the replication cycle, where virions adhere to the surface of host cells through specific interactions between viral surface proteins and host cell receptors. This process is highly selective, determining the virus's host range and tissue tropism. For instance, the spike protein of SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) receptor on human respiratory epithelial cells, facilitating infection primarily in the lungs and upper airways. Similarly, the envelope glycoprotein gp120 of HIV-1 interacts with the CD4 receptor on T lymphocytes and macrophages, a key factor in its targeting of the immune system. Receptor specificity arises from the structural complementarity between viral ligands and host receptors, often involving electrostatic and hydrophobic interactions that mimic natural ligand-receptor bindings. Following attachment, viral entry into the host cell cytoplasm occurs via several mechanisms, each adapted to the virus's structure and the host membrane properties. Enveloped viruses commonly employ membrane fusion, where viral fusion proteins mediate the merging of viral and host membranes, driven by conformational changes triggered by low pH or receptor binding. The hemagglutinin (HA) protein of influenza A virus, for example, undergoes a pH-dependent refolding in the endosome to insert a fusion peptide into the host membrane, enabling viral content release. Non-enveloped viruses, lacking a lipid envelope, often rely on endocytosis followed by endosomal membrane disruption or direct genome injection. Poliovirus, a non-enveloped enterovirus, enters via clathrin-mediated endocytosis and utilizes its capsid proteins to form a pore in the endosomal membrane, influenced by the acidic environment. Energy-dependent processes like endocytosis require ATP hydrolysis, while some bacteriophages use direct penetration without cellular energy input. The presence of an envelope significantly influences entry strategies: enveloped viruses can fuse directly at the plasma membrane or in endosomes, leveraging their lipid bilayer for efficient merging, whereas non-enveloped viruses must breach the host membrane through lytic or pore-forming mechanisms.00178-0) Factors such as receptor density, membrane fluidity, and environmental pH modulate these pathways, with many viruses exploiting endosomal acidification for activation. This entry phase culminates in the virion's contents reaching the cytoplasm, setting the stage for subsequent uncoating.

Uncoating

Uncoating refers to the disassembly of the viral capsid or envelope, which releases the viral genome into the host cell's appropriate compartment for subsequent replication. This step follows viral entry and is essential for making the genome accessible to host machinery. The process can be partial, involving initial exposure of the genome while retaining some capsid integrity, or complete, leading to full capsid breakdown.^[20] Uncoating is typically triggered by host cell conditions encountered after entry, such as acidification in endosomes or exposure to proteolytic enzymes. For instance, in influenza A virus, low pH within late endosomes (around 5.0–6.0) protonates the M2 ion channel, facilitating influx that dissociates the M1 matrix protein shell and releases viral ribonucleoprotein complexes (vRNPs) into the cytoplasm.^[21] Similarly, host proteases like cathepsins B and L mediate uncoating in non-enveloped adeno-associated viruses (AAV2 and AAV8) by cleaving capsid proteins such as VP3, generating specific fragments (e.g., 35 and 27 kDa for AAV8) without fully disrupting the capsid structure, thereby priming the genome for nuclear delivery.^[22] These triggers ensure uncoating occurs in a controlled, spatiotemporal manner, often 30–90 minutes post-infection for influenza.^[21] Mechanisms of uncoating involve conformational rearrangements in viral proteins, proteolytic processing, or interactions with cellular components. Conformational changes, induced by receptor binding or pH shifts, alter capsid stability; in poliovirus, a non-enveloped picornavirus, binding to the CD155 receptor catalyzes a transition from a metastable state, externalizing the amphipathic helix of VP1 for membrane insertion and RNA release in the cytoplasm. Proteolytic cleavage by endosomal cathepsins or other enzymes exposes hidden domains, as seen in AAV where cleavage sites on VP3 facilitate partial disassembly.^[22] In some cases, ribosomal proteins may contribute to disruption, though this is less common. Variations exist between virus types: non-enveloped viruses like poliovirus undergo cytoplasmic uncoating shortly after endocytosis, while enveloped viruses such as herpes simplex virus (HSV-1) transport intact capsids to the nucleus, where docking at nuclear pore complexes triggers genome ejection into the nucleoplasm without full capsid dissolution. Successful uncoating enables genome accessibility for transcription and replication; for example, vRNP release in influenza allows nuclear import and initiation of viral RNA synthesis, while failure—due to pH neutralization or protease inhibition—results in trapped virions and abortive infection.^[21][20] In HSV-1, nuclear genome delivery is critical for immediate-early gene expression, and defects in pore binding halt the cycle. These outcomes underscore uncoating's role as a key regulatory checkpoint in viral replication.

Genome replication and gene expression

Once the viral genome is released into the host cell following uncoating, genome replication commences to produce multiple copies necessary for progeny virion production. This process relies on either host-derived or virus-encoded polymerases, depending on the nature of the viral nucleic acid. For DNA-containing viruses, replication proceeds via semi-conservative DNA synthesis, primarily utilizing the host cell's DNA-dependent DNA polymerases within the nucleus, although some viruses encode their own polymerases to enhance efficiency or evade restrictions. In contrast, RNA viruses depend entirely on virally encoded RNA-dependent RNA polymerases (RdRps) to synthesize full-length complementary RNA strands from the template genome, occurring in cytoplasmic compartments to avoid nuclear surveillance mechanisms. These polymerases exhibit low fidelity compared to host enzymes, enabling rapid evolution but increasing mutation rates during replication. Gene expression in viruses is tightly coordinated with genome replication to ensure the production of non-structural proteins for replication and structural components for assembly. Transcription generates viral mRNA using host or viral machinery: DNA viruses typically hijack the host RNA polymerase II in the nucleus to transcribe genes from their DNA genome, producing capped and polyadenylated mRNAs compatible with host processing. RNA viruses employ their RdRps for transcription, directly synthesizing mRNA from the genomic RNA template without relying on host nuclear transcription factors. These mRNAs are then translated by host ribosomes in the cytoplasm, often exploiting cellular translation initiation factors; viruses may enhance this through internal ribosome entry sites (IRES) or ribosomal frameshifting to maximize protein output from compact genomes. The reliance on host ribosomes underscores viruses as obligate parasites, as they lack their own translational apparatus.^[3] A key feature of viral gene expression is temporal regulation, dividing genes into early and late categories to orchestrate the replication cycle. Early genes, transcribed soon after entry, encode enzymes for genome replication and modulation of host defenses, such as polymerases and transcription factors, allowing subsequent amplification of the viral genome. Late genes, expressed after replication initiates, primarily code for structural proteins like capsid components, ensuring resources are allocated efficiently. This cascade is achieved through promoter strength, chromatin-like modifications on viral DNA, or replication-dependent derepression, preventing premature production of assembly proteins. In RNA viruses, replication and gene expression are often coupled, as replicative intermediates (antigenomic RNAs) serve dual roles as templates for new genomes and as platforms for mRNA synthesis, streamlining the process in the absence of a nucleus. Host cells mount defenses against these processes to limit viral propagation, including RNA interference (RNAi), which targets viral double-stranded RNA intermediates generated during replication. In RNAi, host Dicer enzymes cleave these dsRNAs into small interfering RNAs (siRNAs) that guide the RNA-induced silencing complex (RISC) to degrade viral mRNAs or inhibit translation, particularly effective against RNA viruses in invertebrates and plants but also detected in mammalian cells under certain conditions. This pathway exemplifies innate antiviral immunity, though viruses counter it with suppressor proteins that disrupt siRNA biogenesis. Such interactions highlight the evolutionary arms race between viral replication strategies and host restrictions.^[23][24]

Assembly and maturation

Viral assembly begins with the formation of the nucleocapsid, where capsid proteins self-assemble around the replicated viral genome to create a protective shell. This process relies on specific interactions between capsid subunits and nucleic acids, often driven by electrostatic forces and protein-protein contacts, ensuring the genome is packaged with precise stoichiometry to maintain structural integrity. For non-enveloped viruses, such as picornaviruses, assembly occurs entirely in the cytoplasm, where protomers form pentamers and hexamers that encapsidate the positive-sense RNA genome, leading to the complete virion. In contrast, many double-stranded DNA viruses, including herpesviruses, initiate nucleocapsid assembly in the host cell nucleus, where major capsid proteins organize into icosahedral structures (T=16 symmetry) around the DNA genome, facilitated by scaffolding proteins that are later removed.^[25] Defects in stoichiometry, such as mismatched protein-to-genome ratios, can result in malformed capsids or empty particles incapable of infection, highlighting the precision required for efficient assembly.^[26] For enveloped viruses, assembly extends to envelopment, where the nucleocapsid acquires a lipid bilayer derived from host membranes. This step typically occurs at intracellular membranes, such as the plasma membrane, endoplasmic reticulum, or trans-Golgi network, involving viral glycoproteins and matrix proteins that anchor the nucleocapsid and mediate budding. In herpesviruses, for instance, after nuclear capsid formation, a primary envelopment happens via budding at the inner nuclear membrane, followed by de-envelopment to release nucleocapsids into the cytoplasm for secondary envelopment at cytoplasmic vesicles.^[25] Similarly, in retroviruses like HIV-1, the Gag polyprotein drives assembly at the plasma membrane, incorporating the genome and envelope proteins through multimerization and lipid interactions. These site-specific processes, whether cytoplasmic, nuclear, or membrane-bound, utilize gene products synthesized earlier in the replication cycle to coordinate component recruitment.^[26] Maturation follows assembly, transforming immature virions into infectious particles through structural rearrangements, often triggered by proteolytic cleavage of precursor proteins. In many viruses, a viral protease processes polyproteins to release functional components and induce conformational changes that stabilize the capsid. For example, in HIV-1, the protease cleaves the Gag polyprotein at multiple sites, reorganizing the lattice into a conical capsid and activating enzymes essential for infectivity.^[27] Herpesviruses undergo similar maturation in the nucleus, where the assemblin protease excises scaffolding proteins, prompting rigid-body rotations and translations of capsid subunits to expand and fortify the structure.^[27] In picornaviruses, maturation involves autocatalytic cleavage of the VP0 protein into VP2 and VP4 during or after assembly, which resolves inter-pentamer interfaces and enhances particle stability without external proteases. These maturation events, whether proteolytic or conformational, are tightly regulated to ensure high-fidelity production of viable virions, with incomplete processing leading to non-infectious forms.^[27]

Release

The release phase of viral replication involves the egress of newly assembled virions from the infected host cell, allowing dissemination to new hosts while often balancing viral yield against host cell viability.^[28] This step is crucial for completing the replication cycle, as it determines the number of infectious progeny released and can influence pathogenesis by either preserving or destroying the host cell.^[29] Viruses employ diverse mechanisms for release, including lysis, budding, and exocytosis, each adapted to the virus's structure and lifecycle strategy.^[28] Lysis is a destructive mechanism primarily used by non-enveloped viruses and bacteriophages, where the host cell membrane is ruptured to liberate virions, often leading to cell death as a byproduct.^[30] In bacteriophage T4, this process is tightly regulated by holins—small membrane proteins that form pores in the cytoplasmic membrane at a programmed time—and endolysins, which are muralytic enzymes that degrade the peptidoglycan cell wall once released through the holin lesions, enabling rapid virion escape.^[31] The timing of lysis is genetically controlled to maximize progeny production, typically occurring after sufficient virions have accumulated, preventing premature release that could reduce yield.^[32] Budding is the predominant release strategy for enveloped viruses, where mature virions acquire their lipid envelope by pushing through and pinching off from a host cell membrane, such as the plasma membrane or internal vesicles, without immediate cell lysis.^[33] This process preserves envelope glycoproteins embedded in the host-derived lipid bilayer, which are essential for subsequent attachment and entry into new cells.^[34] Enveloped viruses often recruit host ESCRT (endosomal sorting complex required for transport) machinery via late-domain motifs in their matrix proteins to facilitate membrane scission and virion separation.^[34] In contrast, non-enveloped viruses rarely bud but may use similar non-lytic pathways in some cases.^[35] Exocytosis represents a less common, non-destructive release mechanism where virions are packaged into host-derived vesicles and secreted via the cellular secretory pathway, applicable to both enveloped and some non-enveloped viruses.^[36] This can involve autophagic processes to form membrane-bound carriers that fuse with the plasma membrane, allowing controlled egress while minimizing immune detection.^[36] Regulation of release timing optimizes viral fitness by synchronizing with assembly completion and progeny accumulation, often through viral proteins that delay host cell death until maximal virion numbers are achieved.^[28] For instance, in poliovirus infection, release coincides with virus-induced apoptosis, where caspase activation leads to cell fragmentation and virion liberation, though the virus suppresses full apoptosis to prolong replication.^[37] Similarly, Ebola virus, an enveloped filovirus, buds continuously from the plasma membrane driven by its VP40 matrix protein, which oligomerizes to deform the membrane and recruit ESCRT components, enabling persistent release without immediate host cell destruction.^[38]

Baltimore classification system

System overview

The Baltimore classification system, proposed by virologist David Baltimore in 1971, organizes viruses into seven distinct classes based on the nature of their genetic material and the mechanisms by which they synthesize messenger RNA (mRNA) for protein translation. The criteria emphasize the type of nucleic acid (DNA or RNA), its strandedness (double- or single-stranded), the sense of the genome relative to mRNA (positive or negative), and whether reverse transcription is involved in the replication process. This framework highlights the diversity in viral genome expression strategies, grouping viruses as follows: Class I (double-stranded DNA viruses), Class II (single-stranded DNA viruses), Class III (double-stranded RNA viruses), Class IV (positive-sense single-stranded RNA viruses), Class V (negative-sense single-stranded RNA viruses), Class VI (single-stranded RNA reverse-transcribing viruses), and Class VII (double-stranded DNA reverse-transcribing viruses). The primary purpose of the Baltimore classification is to predict key aspects of viral replication, including the required polymerases and intermediates, which in turn informs host range specificity and potential targets for antiviral therapies. For instance, it elucidates how viruses exploit or bypass host cellular machinery, such as DNA-dependent RNA polymerases for DNA viruses or RNA-dependent RNA polymerases for many RNA viruses, to generate functional mRNA. A foundational principle across all classes is that viruses must ultimately produce positive-sense mRNA compatible with host ribosomes for translation into viral proteins, though the pathways to achieve this vary significantly—ranging from direct use of the genome as mRNA in Class IV to multi-step transcription and reverse transcription in Classes VI and VII. These differences underscore the evolutionary adaptations viruses have developed to replicate within diverse host environments, from bacteria to humans. While the system excels at delineating genome replication and gene expression, it has notable limitations, such as its exclusion of post-transcriptional stages like viral assembly and release, which are universal across virus classes but mechanistically diverse. Originally focused on animal viruses, Despite these constraints, its enduring utility lies in providing a predictive lens for viral diversity without relying on phenotypic traits like morphology or host specificity.

Class I: dsDNA viruses

Class I viruses possess double-stranded DNA (dsDNA) genomes, which serve as templates for replication and transcription in a manner analogous to cellular DNA processes.^[39] These genomes are typically linear or circular, ranging in size from 5 to 250 kilobases (kb), accommodating genes for structural proteins, enzymes, and regulatory elements.^[40] For instance, adenoviruses feature linear dsDNA genomes of approximately 36 kb, while herpesviruses have larger linear genomes spanning 125–240 kb.^[1] Unlike other viral classes, Class I viruses do not require reverse transcriptase for genome replication, relying instead on DNA-dependent DNA polymerases.^[40] Replication of Class I viral genomes predominantly occurs in the host cell nucleus, exploiting the cellular DNA synthesis machinery during the S-phase of the cell cycle.^[1] Smaller viruses, such as polyomaviruses and papillomaviruses, utilize host DNA polymerases for bidirectional theta-mode replication from a single origin, producing circular progeny genomes.^[40] Larger viruses like herpesviruses and adenoviruses encode their own DNA polymerases and often employ rolling-circle replication to generate linear concatemers, which are later processed into unit-length genomes.^[39] Poxviruses represent a notable exception, replicating their linear dsDNA genomes entirely in the cytoplasm using virally encoded enzymes, independent of host nuclear processes.^[1] Transcription in Class I viruses is mediated by the host's RNA polymerase II, which recognizes viral promoters structurally similar to eukaryotic promoters, enabling temporal regulation of early (regulatory) and late (structural) gene expression.^[40] These transcripts are capped, polyadenylated, and spliced in the nucleus before export to the cytoplasm for translation.^[39] In poxviruses, however, a virally encoded multi-subunit RNA polymerase performs transcription within cytoplasmic factories.^[1] Prominent examples include herpesviruses, which establish latency by maintaining their dsDNA as episomes in the host nucleus, reactivating under stress to produce lytic progeny.^[39] Adenoviruses replicate nuclearly and induce cell cycle progression to favor S-phase.^[1] Bacteriophages such as lambda exemplify lysogeny in prokaryotic hosts, where the linear dsDNA genome circularizes and integrates into the bacterial chromosome via site-specific recombination, persisting without replication until induction triggers lytic growth.^[40]

Class II: ssDNA viruses

Class II viruses, also known as single-stranded DNA (ssDNA) viruses, possess genomes consisting of a single strand of DNA, which can be either circular or linear, with sizes typically ranging from 1.7 to 6 kb.^[41] Most members, such as those in the Geminiviridae and Circoviridae families, feature circular ssDNA genomes that are generally of positive sense, allowing for direct use as a template after conversion, while Parvoviridae exhibit linear ssDNA genomes that may package either positive or negative strands and are flanked by terminal hairpin structures to facilitate replication initiation.^[42] These small genomes encode a limited number of proteins, usually including a replication initiator protein and capsid components, reflecting their heavy reliance on host cellular machinery.^[41] Upon entry into the host cell nucleus, the ssDNA genome is converted to a double-stranded DNA (dsDNA) replicative form by host DNA polymerase, providing a template for both replication and transcription.^[42] For circular ssDNA viruses like geminiviruses, replication proceeds via a rolling-circle mechanism, where the virus-encoded replication-associated protein (Rep) nicks the DNA at the origin of replication and recruits host polymerases to generate multimeric dsDNA intermediates, which are then processed into unit-length progeny ssDNA for packaging.^[42] In contrast, linear ssDNA viruses such as parvoviruses employ a rolling-hairpin replication strategy, utilizing the terminal hairpins to fold back and prime continuous synthesis, again dependent on host DNA replication factors like polymerase δ and PCNA.^[43] This process occurs exclusively in the nucleus and often requires the host cell to be in S-phase to access replication machinery.^[43] Transcription of viral genes occurs from the dsDNA intermediate using the host's RNA polymerase II machinery, producing mRNAs that are often bidirectional or ambisense to maximize coding capacity from the compact genome.^[41] For instance, in parvoviruses, a single promoter drives transcription of a polycistronic pre-mRNA that undergoes alternative splicing to generate multiple transcripts encoding non-structural proteins like NS1 (involved in replication) and structural capsid proteins VP1 and VP2.^[43] Representative examples include human parvovirus B19, a Parvoviridae member with a 5.6 kb linear ssDNA genome that replicates in erythroid progenitor cells during S-phase, causing conditions like erythema infectiosum and transient aplastic crisis due to its dependence on host cell division.^[43] In plants, geminiviruses such as those in the genus Begomovirus (e.g., tomato yellow leaf curl virus) feature circular ssDNA genomes of about 2.7 kb and replicate in leaf mesophyll cells, leading to diseases like leaf curling and stunting by hijacking host replication factors without inducing cell division.^[42] These viruses face significant constraints due to their small genome size (~5 kb on average), limiting autonomous replication and necessitating exploitation of host S-phase factors, which restricts infection to dividing or manipulable cells and increases vulnerability to host defenses and environmental damage like UV radiation.^[41]

Class III: dsRNA viruses

Class III viruses, also known as double-stranded RNA (dsRNA) viruses, belong to the Baltimore classification system and are characterized by genomes consisting of segmented dsRNA. The primary family within this class is Reoviridae, which encompasses genera such as Orthoreovirus, Rotavirus, and Orbivirus. These viruses possess 10 to 12 linear dsRNA segments, with total genome sizes ranging from approximately 16 to 27 kilobases (kb).^[44] For instance, mammalian orthoreoviruses have 10 segments totaling about 23.7 kb, while rotaviruses feature 11 segments amounting to roughly 18.5 kb.^[45][46] The segmented nature allows for genetic reassortment during co-infection, contributing to viral diversity and evolution.^[47] Replication of dsRNA viruses occurs entirely in the cytoplasm, independent of host nuclear machinery, and relies exclusively on the virus-encoded RNA-dependent RNA polymerase (RdRp). Upon entry into the host cell, the viral capsid partially uncoats to release transcriptionally active subviral cores containing the dsRNA genome and associated RdRp complexes, such as VP1 in rotaviruses. Transcription proceeds via a conservative mechanism, where the RdRp synthesizes positive-sense (+)-single-stranded RNA (ssRNA) mRNAs directly from the negative-sense (-) strand of the dsRNA template without fully unwinding or displacing the parental dsRNA duplex.^[48][49] These viral mRNAs are capped at the 5' end by a viral guanylyltransferase but lack polyadenylation at the 3' end, featuring instead a conserved tetranucleotide sequence that terminates transcription.^[50] The +ssRNA transcripts are then extruded through channels in the core shell for translation into viral proteins by host ribosomes. Genome replication follows a semi-conservative strategy within cytoplasmic viral factories—discrete inclusion bodies formed by viral non-structural proteins like NSP2 in rotaviruses—where +ssRNA serves as templates for synthesizing full-length -ssRNA, which anneals with the template to form progeny dsRNA segments.^[51] This process is tightly coupled with packaging, as nascent dsRNA segments are incorporated into assembling core particles.^[52] Prominent examples include rotavirus A, a major cause of severe dehydrating diarrhea in infants and young children worldwide, and mammalian reoviruses, which are typically asymptomatic in humans but serve as models for studying dsRNA virus replication.^[46] In reovirus infection, viral factories form perinuclear aggregates that concentrate replication machinery, facilitating efficient dsRNA synthesis and virion assembly without nuclear involvement.^[53] A distinctive feature of Class III viruses is their sequestration of dsRNA within protective core structures throughout the replication cycle, which shields the genome from host Dicer-mediated RNA interference and innate immune sensors.^[54] This packaging strategy, combined with the cytoplasmic localization, enables robust replication in diverse host cells while minimizing antiviral responses.^[55]

Class IV: (+)ssRNA viruses

Class IV viruses, also known as positive-sense single-stranded RNA (+ssRNA) viruses, possess a linear, non-segmented RNA genome that directly serves as messenger RNA (mRNA) for protein synthesis upon infection.^[56] These genomes typically range from 7 to 30 kilobases (kb) in length, with the majority falling between 8 and 12 kb for families like Picornaviridae and Flaviviridae, while larger genomes up to approximately 31 kb are found in Coronaviridae.^[57] The 5' end of the genomic RNA is usually capped with a 7-methylguanosine structure, and the 3' end is polyadenylated, mimicking eukaryotic mRNA to facilitate host ribosomal recognition and translation.^[56] However, exceptions exist, such as in picornaviruses, where a viral protein (VPg) is covalently linked to the 5' end instead of a cap.^[56] Upon entry into the host cell cytoplasm, the +ssRNA genome is immediately translated by host ribosomes via a cap-dependent scanning mechanism from the 5' end to initiate viral protein synthesis.^[58] In certain families like Picornaviridae, translation is cap-independent and relies on an internal ribosome entry site (IRES) in the 5' untranslated region (UTR), allowing direct recruitment of the 40S ribosomal subunit even under cellular stress conditions that inhibit cap-dependent translation.^[59] This initial translation produces viral proteins, including the RNA-dependent RNA polymerase (RdRp), which is essential for genome replication and shared in function with RdRps from other RNA virus classes.^[58] The RdRp then uses the genomic RNA as a template to synthesize a complementary negative-sense (-) RNA strand in membrane-bound replication complexes within the cytoplasm, avoiding host antiviral responses.^[56] The (-) RNA intermediate serves as a template for producing new +ssRNA genomes and, in some cases, subgenomic mRNAs for expressing downstream genes.^[60] For viruses like coronaviruses, subgenomic mRNAs are generated through a unique discontinuous transcription mechanism, where the RdRp pauses at transcription-regulatory sequences (TRS) and jumps to the 5' leader sequence, resulting in a nested set of 5'-capped, polyadenylated mRNAs that are 3'-coterminal with the genome.^[60] This strategy enables efficient expression of structural and accessory proteins from polycistronic genomes. Replication occurs entirely in the cytoplasm, with no nuclear involvement.^[56] Representative examples include coronaviruses, such as SARS-CoV-2, which exhibit recombination hotspots particularly around the spike gene region due to template-switching during replication, contributing to viral evolution and host adaptation.^[61] Flaviviruses like Zika virus replicate via a similar RdRp-mediated process but lack proofreading activity in their polymerase, leading to high mutation rates of approximately 10^{-4} to 10^{-5} substitutions per site per replication cycle, fostering genetic diversity and quasispecies formation.^[62] These features underscore the adaptability of Class IV viruses, which comprise a diverse group responsible for diseases ranging from common colds to severe epidemics.^[56]

Class V: (-)ssRNA viruses

Class V viruses, also known as negative-sense single-stranded RNA (-ssRNA) viruses, possess genomes that are complementary to messenger RNA (mRNA) and thus cannot be directly translated by host ribosomes upon entry into the cell.^[63] These viruses belong to several families within the order Mononegavirales (non-segmented) and other groups like Orthomyxoviridae and Bunyaviridae (segmented), with linear genomes ranging from 8 to 20 kilobases (kb) in length.^[64] Non-segmented genomes, such as those of rhabdoviruses like rabies virus, consist of a single continuous -ssRNA strand, while segmented genomes, exemplified by influenza A virus with eight segments totaling approximately 13.5 kb, allow for independent replication and packaging of gene modules.^[63] The viral genome is invariably encapsidated by nucleoproteins (N or NP) into helical or circular ribonucleoprotein (RNP) complexes, which protect the RNA and serve as templates for replication and transcription.^[65] Replication and transcription in Class V viruses are mediated by the viral RNA-dependent RNA polymerase (RdRp), a large multifunctional complex (typically 250–450 kDa) comprising the polymerase (L protein) and accessory phosphoprotein (P or VP35), always pre-packaged within the incoming virion's RNP to initiate infection.^[63] Upon entry, the RNP is released into the cytoplasm or nucleus, where the RdRp first transcribes the -ssRNA genome into positive-sense (+)-mRNA through a primary transcription step, producing monocistronic mRNAs that are capped, polyadenylated, and exported for translation into viral proteins.^[65] Transcription occurs in a sequential, stop-start manner from 3' to 5' along the genome, with gene-end signals triggering polymerase stuttering for poly(A) tails.^[66] Capping of these mRNAs is achieved via cap-snatching in segmented viruses like influenza (Orthomyxoviridae) and bunyaviruses (Bunyaviridae), where the RdRp's endonuclease domain cleaves 5' caps from host pre-mRNAs to prime viral transcription, enhancing stability and translation efficiency.^[67] In contrast, non-segmented viruses such as rabies (Rhabdoviridae) and Ebola (Filoviridae) initiate transcription de novo without cap-snatching, relying on internal priming mechanisms.^[63] Replication follows, switching the RdRp mode to synthesize full-length antigenomic +RNA intermediates, which are then used to produce excess genomic -ssRNA; this process requires accumulating nucleoproteins to encapsidate nascent RNA and prevent degradation.^[65] The RdRp operates with an error rate of approximately 10^{-4} to 10^{-5} mutations per nucleotide, contributing to genetic diversity despite lacking proofreading activity.^[68] Site of replication varies: most -ssRNA viruses, including rabies and Ebola, replicate in the cytoplasm within RNP factories, while orthomyxoviruses like influenza replicate in the nucleus, necessitating nuclear import of RNPs via nuclear localization signals and export of mRNAs through host pathways.^[63] This nuclear phase in influenza involves cap-snatching from nuclear host transcripts and integration with host splicing machinery for some segments.^[64] Examples illustrate these features: influenza virus exports newly synthesized RNPs from the nucleus using the M1 protein and NEP for packaging; rabies virus forms cytoplasmic inclusion bodies for efficient replication; and Ebola virus, a filovirus, assembles RNPs in the cytoplasm with VP35 aiding polymerase processivity.^[66] These mechanisms ensure coordinated genome amplification and gene expression tailored to the viral lifecycle.^[65]

Class VI: ssRNA-RT viruses

Class VI viruses, also known as ssRNA-RT viruses or retroviruses, possess a positive-sense single-stranded RNA genome that is replicated through a DNA intermediate using reverse transcriptase.^[69] These viruses package two identical copies of their genomic RNA, forming a diploid structure that facilitates genetic recombination during replication.^[70] The genome typically ranges from 7 to 11 kilobases in length and is flanked by long terminal repeats (LTRs) at both ends, which consist of three regions (U3, R, and U5) essential for integration and gene expression.^[70] The LTRs contain promoter and enhancer sequences that drive transcription once integrated into the host genome.^[71] Replication begins in the cytoplasm shortly after viral entry, where the viral reverse transcriptase (RT) enzyme, encoded by the pol gene, initiates the conversion of the RNA genome into double-stranded DNA (dsDNA).^[70] RT, a heterodimeric protein with both DNA polymerase and RNase H activities, uses a host tRNA as a primer to synthesize the minus-strand DNA from the RNA template, followed by degradation of the RNA via RNase H.^[70] This process involves two strand transfers: the first mediated by repeat (R) sequences at the genome ends, and the second using polypurine tracts (PPTs) to initiate plus-strand synthesis, ultimately yielding a linear dsDNA molecule with LTRs at both termini.^[70] The pre-integration complex, including the dsDNA, RT, and integrase, is then transported to the nucleus, where the viral integrase enzyme catalyzes the insertion of the dsDNA into the host chromosomal DNA, forming a provirus that can remain latent.^[69] Once integrated, the provirus serves as a template for transcription by the host's RNA polymerase II, initiated from the 5' LTR promoter.^[71] This produces full-length primary transcripts that function both as genomic RNA for new virions and as precursors for spliced messenger RNAs (mRNAs) encoding viral proteins such as envelope (Env) glycoproteins.^[71] The viral mRNAs acquire a 5' cap and 3' poly(A) tail through host cellular machinery, utilizing signals in the LTR's R and U3 regions for polyadenylation.^[71] Unspliced full-length transcripts are exported to the cytoplasm for translation of Gag and Gag-Pol polyproteins and packaging into progeny virions, while spliced transcripts produce accessory proteins like Tat and Rev in lentiviruses.^[71] Prominent examples include human immunodeficiency virus (HIV), a lentivirus that targets CD4+ T cells via its envelope glycoprotein binding to CD4 and co-receptors, leading to acquired immunodeficiency syndrome (AIDS), and human T-lymphotropic virus (HTLV-1), an oncogenic deltaretrovirus associated with adult T-cell leukemia/lymphoma through proviral integration and Tax protein-mediated oncogenesis.^[72] Both exemplify the class's capacity for latency, as the integrated provirus persists in the host genome without immediate replication.^[69] A distinctive feature of these viruses is the error-prone nature of RT, which lacks 3'–5' exonuclease proofreading activity and exhibits an in vitro error rate of approximately 10^{-4} mutations per nucleotide, driving rapid genetic diversification and immune evasion, particularly in HIV.^[70] This high mutation rate, combined with the diploid genome, enables recombination and adaptation.^[70]

Class VII: dsDNA-RT viruses

Class VII viruses, also known as dsDNA-RT viruses, are characterized by a partially double-stranded DNA genome that replicates through a reverse transcription intermediate, distinguishing them from other DNA viruses by their reliance on an RNA template for genome amplification.^[1] The genome typically ranges from 3 to 8 kb and features gaps in the double-stranded structure, often forming a relaxed circular conformation in hepadnaviruses, with a complete minus strand covalently linked to the viral polymerase and an incomplete plus strand primed by RNA.^[73] This gapped DNA is repaired in the host nucleus to form a covalently closed circular DNA (cccDNA) episome, which serves as the template for transcription but does not typically integrate into the host genome.^[74] Replication occurs via protein-primed reverse transcription, where the viral reverse transcriptase (RT), part of the polymerase protein (P), initiates DNA synthesis using a tyrosine residue as a primer on the pregenomic RNA (pgRNA) template within cytoplasmic nucleocapsids.^[73] Transcription of the pgRNA and other viral mRNAs is carried out by the host RNA polymerase II in the nucleus from the cccDNA, producing a terminally redundant pgRNA (~3.2 kb in hepatitis B virus) that is packaged into core particles along with the P protein.^[74] The RT then synthesizes the minus-strand DNA from the pgRNA, followed by partial plus-strand synthesis, yielding the relaxed circular DNA (rcDNA) genome; this process requires host chaperones like Hsp90 for P activation and occurs entirely within the capsid to ensure fidelity.^[1] Unlike semiconservative DNA replication in Class I viruses, this RT-mediated amplification allows for high-fidelity genome production despite the RNA intermediate.^[73] A prominent example is the hepatitis B virus (HBV) from the Hepadnaviridae family, which causes acute and chronic liver infections leading to hepatitis, cirrhosis, and hepatocellular carcinoma in chronic cases.^[1] HBV's persistence stems from the stable cccDNA pool in hepatocytes, with a half-life of 30-60 days, enabling lifelong infection even under immune pressure without genomic integration.^[74] This nuclear localization of cccDNA mirrors aspects of Class I viral transcription but uniquely couples it to cytoplasmic RT for progeny genome production.^[73] The absence of routine integration and the specialized RT mechanism underscore Class VII viruses' adaptation for chronicity in vertebrate hosts.^[1]