Virus Replication

Why replication is central

A virion is inert. It carries no ribosomes, generates no energy, and on its own can do nothing. Everything that makes a virus a virus happens after it enters a cell, and all of it is borrowed: the host supplies the protein factory, the building blocks and the power. The virus brings only information.

That dependence sets the single problem from which the whole of replication follows. Host ribosomes translate only messenger RNA of the positive sense. Whatever a virus carries as its genome (DNA or RNA, single- or double-stranded, positive- or negative-sense), it must somehow turn that genome into mRNA the cell will read, then copy the genome itself for the next generation. The economy of viruses is that they rewire a cell into a factory for making more of themselves using only a handful of genes.

The Baltimore classification

David Baltimore’s insight in 1971 was to group all viruses not by what they look like but by how their genome reaches mRNA. Because the ribosome reads only positive-sense mRNA, each kind of genome faces a different journey to get there, and that journey is the most fundamental thing about a virus. The scheme began with six classes and now has seven:

Class	Genome	Route to mRNA and replication	Examples
I	dsDNA	Transcribed by host RNA polymerase in the nucleus (poxviruses carry their own polymerase and stay in the cytoplasm)	Herpesviruses, adenoviruses, poxviruses, papillomaviruses
II	ssDNA	Converted to a double-stranded intermediate, then transcribed	Parvoviruses (including AAV)
III	dsRNA	Must carry its own polymerase; transcribes mRNA from within the core	Reoviruses (rotavirus)
IV	(+)ssRNA	Genome is mRNA: translated directly on entry; replicated via a (−)-sense template	Picornaviruses, flaviviruses, coronaviruses, togaviruses
V	(−)ssRNA	Must carry its own polymerase to transcribe (+)-sense mRNA first	Influenza, paramyxoviruses, rhabdoviruses, filoviruses
VI	(+)ssRNA-RT	Reverse-transcribed to DNA, integrated as a provirus, then transcribed by the host	Retroviruses (HIV)
VII	dsDNA-RT	Transcribed to an RNA pregenome, then reverse-transcribed back to DNA	Hepadnaviruses (hepatitis B)

A host cell has no enzyme that can copy RNA from an RNA template or translate a negative-sense strand. Negative-sense RNA viruses (rabies, the filoviruses, influenza) therefore cannot have their genome read by a ribosome on entry: they must carry their own polymerase inside the virion to transcribe positive-sense mRNA first. The double-stranded RNA viruses (the reoviruses), equally unreadable, package their polymerase for the same reason. Positive-sense RNA viruses (picornaviruses, flaviviruses, coronaviruses) face no such barrier: their genome is mRNA, translated the moment it arrives, with the polymerase made on the spot.

The replication cycle

Infect a synchronised culture at high multiplicity and follow the virus over time and a characteristic one-step growth curve appears. Immediately after entry the inoculated virus seems to vanish: no infectious particles can be recovered even by breaking the cells open. This is the eclipse period, the hours during which the genome has been uncoated and is directing synthesis but no finished progeny yet exist. Then new virions appear and titres climb steeply.

Every virus runs the same fixed sequence, and that sequence doubles as the map of where antiviral drugs act: almost every step has a licensed inhibitor, and knowing the step explains the drug.

#	Step	What happens	Clinical correlate
1	Attachment	Viral ligand binds host receptor (± co-receptor); sets host range and tropism	Entry inhibitors (maraviroc → CCR5); neutralising antibodies
2	Penetration	Genome crosses the plasma membrane: pH-dependent endocytosis or pH-independent fusion	Entry route dictates efficacy (chloroquine failed against surface-route SARS-CoV-2); enfuvirtide blocks HIV fusion
3	Uncoating	Capsid shed or rearranged, releasing the genome into cytoplasm or nucleus	Amantadine blocks the influenza A M2 channel needed for uncoating
4	Transcription & translation	Viral mRNA commandeers host ribosomes; early (regulatory/enzymatic) then late (structural) proteins	Protease inhibitors (HIV “-navir”, HCV “-previr”, SARS-CoV-2 nirmatrelvir)
5	Genome replication	Genome amplified by DNA polymerase, RdRp or reverse transcriptase, inside membranous factories	RT, polymerase and integrase inhibitors: SA first-line ART (tenofovir, lamivudine, dolutegravir), aciclovir, ganciclovir, sofosbuvir/velpatasvir
6	Assembly	Proteins and genomes self-assemble; maturation cleavages confer infectivity	Capsid and packaging inhibitors (lenacapavir; letermovir for CMV); protease inhibitors leave particles immature
7	Release	Lysis (non-enveloped) or budding (enveloped, via ESCRT); some exit in host vesicles	Neuraminidase inhibitors (oseltamivir, zanamivir) block influenza release

1. Attachment (adsorption)

The cycle begins with a specific binding event at the cell surface. A protein on the virus, the ligand, binds a molecule on the host cell, the receptor; many viruses then engage a second co-receptor. Which cells display the receptor is a large part of why a virus infects the tissues it does, its tropism, and therefore what disease it causes.

Influenza haemagglutinin binds sialic acid, and whether a strain prefers the α2,6-linked form (human upper airway) or the α2,3-linked form (avian gut, deep human lung) helps determine which species it spreads in, a feature watched closely in pandemic surveillance. HIV-1 binds in sequence: gp120 first holds CD4 (hence its tropism for CD4 T-helper cells and macrophages), exposing a site that then engages a chemokine co-receptor, CCR5 or CXCR4.

Clinical correlate. This step is blocked by entry inhibitors: maraviroc occupies CCR5 so HIV cannot reach it, and people who inherit a non-functional CCR5 allele (CCR5-Δ32) are largely protected for the same reason. Neutralising antibodies also act here.

2. Penetration (entry)

The genome must now cross the membrane. Most viruses are internalised by endocytosis, the constitutive process by which a cell takes patches of its surface into a membrane vesicle, the endosome; the endosome acidifies as it matures, and for many enveloped viruses that falling pH springs the fusion protein, which merges the viral envelope with the endosomal membrane and delivers the genome to the cytoplasm. Others fuse directly through the plasma membrane at neutral pH (paramyxoviruses, HIV). Non-enveloped viruses, with no membrane to merge, form a pore and pass their genome through it. The route can depend on the cell: SARS-CoV-2 fuses at the surface where the protease TMPRSS2 is present to cleave its spike, but is otherwise taken into the endosome and cleaved there by cathepsin L.

Clinical correlate. Entry route determines drug efficacy. Chloroquine, which de-acidifies endosomes, blocked SARS-CoV-2 in cultured kidney cells (the endosomal route) but failed in patients, whose airway cells use the surface route. Enfuvirtide blocks HIV by jamming its fusion protein partway through its conformational change.

3. Uncoating

The capsid is shed or conformationally rearranged, releasing the genome into the compartment where it will be copied: the cytoplasm for most RNA viruses, the nucleus for most DNA viruses and the retroviruses. For some viruses penetration and uncoating are a single coordinated event; for others uncoating is staged, completed only once the genome reaches its destination.

Clinical correlate. Amantadine and rimantadine block the influenza A M2 ion channel, preventing the acidification of the virion interior that uncoating requires. Near-universal resistance has retired them from use, but they remain the classic example of an uncoating inhibitor.

4. Transcription and translation

Viral genes are expressed on a temporal programme: early genes encode regulatory proteins and the enzymes of genome replication, together with proteins that blunt host defences; late genes encode the structural proteins, produced in bulk.

A eukaryotic ribosome translates only the first open reading frame of a polycistronic message (several genes in a row) and then stops, so a virus cannot simply present its genome to be read wholesale. The most striking solution is to translate the entire coding region as one polyprotein and cleave it into functional units with a viral protease (an enzyme that cuts proteins); others splice or segment their mRNAs, use ribosomal frameshifting, or carry an internal ribosome entry site (IRES) that lets translation begin within a message rather than only at its start.

Clinical correlate. The polyprotein strategy opens a large therapeutic window, because a protease inhibitor leaves the polyprotein uncut and the progeny non-infectious. This is the basis of the HIV “-navir” drugs (such as ritonavir), the hepatitis C “-previr” drugs, and nirmatrelvir (the active component of Paxlovid) against SARS-CoV-2.

5. Genome replication

The genome is amplified. DNA viruses copy theirs in the nucleus by a replication fork or by strand displacement, using a DNA polymerase. RNA viruses confront a problem the host cannot solve: no cellular enzyme copies RNA from an RNA template, so each must carry or encode an RNA-dependent RNA polymerase (RdRp) (the retroviruses and hepadnaviruses instead use reverse transcriptase).

Replication does not proceed free in the cytoplasm but is walled off inside dedicated replication organelles, membranous factories remodelled from host membranes: positive-sense RNA viruses fold pockets from the endoplasmic reticulum, the reoviruses build dense viroplasms, and negative-sense RNA viruses gather their machinery into liquid-like inclusion bodies. Assembly of progeny often begins in or beside these same compartments.

The factories concentrate the machinery and, just as importantly, conceal the virus: double-stranded RNA is a molecular signature of viral copying that the cytoplasmic sensors RIG-I and MDA-5 detect to trigger interferon, the cell’s principal antiviral alarm. Sequestering replication keeps that signal out of view, and the same pressure shapes genome strategy, positive-sense viruses holding their double-stranded intermediates to a minimum and negative-sense viruses keeping their genomes sheathed in nucleoprotein so they never present naked RNA.

Clinical correlate. With no host counterpart, the viral polymerases are the most heavily drugged step of all, and they carry the backbone of South African antiretroviral therapy. Tenofovir and lamivudine are nucleos(t)ide analogues that terminate reverse transcription, nevirapine a non-nucleoside inhibitor that jams the same enzyme, and dolutegravir (with the long-acting cabotegravir) blocks the integrase that splices the reverse-transcribed DNA into the host genome.

The same logic targets DNA polymerases: aciclovir and ganciclovir, each switched on only by a viral kinase before halting the viral DNA polymerase of herpes simplex/VZV and of CMV respectively, and the hepatitis C cure sofosbuvir/velpatasvir, a polymerase inhibitor paired with an NS5A inhibitor that collapses the replication complex. This error-prone copying also drives drug resistance and antigenic drift.

6. Assembly (maturation)

Newly synthesised structural proteins and genomes self-assemble into nucleocapsids, frequently within or beside the replication factories. Many viruses are not infectious as first assembled and must undergo maturation, typically a proteolytic cleavage that locks the particle into its functional form, as in the protease-driven maturation of HIV or the final capsid cleavage of the picornaviruses.

Clinical correlate. Maturation is targetable: by leaving the polyprotein uncut, the HIV protease inhibitors yield assembled but immature, non-infectious virions, so this step and the gene-expression step are defeated by the same drugs.

7. Release (egress)

Non-enveloped viruses are classically freed when the cell bursts (lysis), while enveloped viruses bud through a membrane studded with their glycoproteins, acquiring their envelope as they leave. The scission that frees a budding particle is performed not by the virus but by the host’s ESCRT machinery, which the virus recruits by displaying short “late-domain” motifs (influenza is the exception, severing itself with its M2 protein).

The clean lysis-versus-budding division is no longer absolute: poliovirus, coxsackievirus, hepatitis A and hepatitis E are now known to exit non-lytically inside host membrane vesicles (quasi-enveloped), and the hepatitis A circulating in blood is entirely cloaked this way, shielded from antibody.

Clinical correlate. Influenza progeny bud out still tethered to the cell by haemagglutinin’s grip on sialic acid; the viral enzyme neuraminidase must cleave those sugars to release them. The neuraminidase inhibitors oseltamivir and zanamivir prevent that cleavage, trapping progeny at the surface and halting spread.

Growing viruses in the laboratory

Viruses replicate only inside living cells, so they must be grown in a host system. Embryonated hens’ eggs, used since the 1930s, are still the workhorse for producing influenza virus and much influenza vaccine. Cell culture transformed the field by making intracellular events visible and infectious virus countable.

Three kinds of culture are used. Primary cells, freshly dissociated from tissue, behave most like the real host but divide only a few times. Diploid cell strains such as the lung fibroblasts WI-38 and MRC-5 are normal cells that senesce after roughly fifty divisions, the Hayflick limit, as their telomeres shorten. Continuous cell lines such as HeLa and BHK-21, derived from tumours or transformed cells, divide indefinitely and so give consistent, convenient stocks, at the cost of having drifted far from any normal tissue.

Infection is often recognised down the microscope by the cytopathic effect, the characteristic rounding, fusion or death of cells, which for some families is distinctive enough to suggest the agent.

Counting viruses

A virus suspension can be quantified physically (counting particles directly, for example by electron microscopy) or biologically (measuring infectivity), and the two rarely agree. The classic biological method is the plaque assay, developed for animal viruses by Dulbecco and Vogt: dilutions of virus are added to a cell monolayer under a semi-solid overlay that stops virus diffusing, and each infectious particle that founds a spreading focus of dead cells produces one countable plaque, so the titre is read in plaque-forming units.

The gap between the two kinds of count is the particle-to-infectivity ratio, often greater than a thousand to one: many particles carry a defective genome, are damaged, or simply fail to start an infection even in a permissive cell, so most never form a plaque. This is also why viral load measured by PCR, which counts genome copies, is not the same as infectious titre: it cannot tell a whole virion from an empty shell.

Generating diversity

Every virus stock is not a single sequence but a swarm of closely related ones, a quasispecies, clustered around a consensus. The width of that swarm depends on the copying enzyme. DNA polymerases proofread and so make few errors; the RNA-dependent RNA polymerases and reverse transcriptases of RNA viruses do not, and make errors roughly ten thousand times more often, which is why RNA viruses evolve so fast and escape immunity and drugs so readily. Diversity is generated further by recombination and, in viruses with segmented genomes, by reassortment, the swapping of whole segments between two viruses in one cell that drives the abrupt antigenic shifts of influenza.

That error rate comes at a price. Above a certain rate a genome can no longer be copied faithfully enough to preserve itself, the error threshold, and this caps RNA genomes at around thirty kilobases. The coronaviruses are the striking exception: they reach that size only because they carry a proofreading exonuclease (nsp14) that no other RNA viruses have. The same fragility is a drug target, since a mutagen such as ribavirin can tip a virus past its threshold into error catastrophe, where the population accumulates lethal mutations and dies out. Diversity is also exploited deliberately: passaging a virus repeatedly in culture selects variants adapted to the dish and attenuated in the host, which is the classical route to a live vaccine strain.

References and recommended reading

• Burrell CJ, Howard CR, Murphy FA. Virus Replication. In: Fenner and White’s Medical Virology, 5th edition. Academic Press / Elsevier; 2017. Chapter 4. The readable backbone for this material: the Baltimore scheme, the replication cycle, cultivation, quantification and genetic diversity.
• Mercer J, Whelan SPJ, et al. Virus Entry and Uncoating; Viral Replication Strategies; Virus Assembly and Maturation. In: Fields Virology, 7th edition, Volume 4: Fundamentals. Wolters Kluwer; 2023. Chapters 4–6. The current, deeper treatment; the source for membranous replication organelles and innate-sensor evasion, the ESCRT scission machinery, non-lytic vesicle release, the error-catastrophe concept with the coronavirus proofreading exception, and the SARS-CoV-2 dual entry route.