Adenovirus Life Cycle: Stages and Key Molecular Events

Adenoviruses are non-enveloped DNA viruses that infect a wide range of hosts, including humans. They cause illnesses ranging from mild respiratory infections to severe diseases in immunocompromised individuals. Their ability to efficiently enter host cells, hijack cellular machinery, and evade immune responses has made them a subject of extensive study.

Understanding the adenovirus life cycle provides insight into its replication strategies and potential therapeutic applications. Each stage involves precise molecular events that enable viral propagation.

Virion Structure

Adenoviruses have an icosahedral capsid composed of 240 hexon trimers and 12 penton base proteins, forming a stable, non-enveloped structure about 90 nm in diameter. The hexon proteins stabilize the capsid, while the penton bases, located at each vertex, mediate host cell attachment. Extending from each penton base is a fiber protein, which determines tropism by facilitating receptor binding. Variations in fiber length and receptor specificity influence infectivity and tissue preference.

Inside the capsid, the viral core houses a linear, double-stranded DNA genome of approximately 26-45 kilobases. This genome is tightly associated with viral proteins, including protein VII, which condenses the DNA, and protein V, which links the genome to the inner capsid. Terminal protein (TP) is covalently attached to the 5’ ends of the DNA, playing a role in replication. These core proteins ensure genome stability and facilitate efficient unpacking upon entry into the host cell.

Transmission And Host Entry

Adenoviruses spread through respiratory droplets, fecal-oral transmission, and contact with contaminated surfaces or bodily fluids. Respiratory serotypes like HAdV-B3 and HAdV-C2 spread via aerosolized droplets, while enteric serotypes such as HAdV-F40 and HAdV-F41 are transmitted through contaminated food or water. Their stability allows them to persist on surfaces for extended periods, increasing the likelihood of indirect transmission.

Upon encountering a host, the virus binds to specific cell surface receptors. Most human adenoviruses use the coxsackievirus and adenovirus receptor (CAR) for attachment, while some, like HAdV-B serotypes, engage CD46. This receptor specificity influences tissue tropism. After attachment, the penton base proteins interact with αv integrins, triggering receptor-mediated endocytosis through clathrin-coated vesicles.

Inside the cell, the virus travels through the endosomal pathway, where pH-dependent capsid changes facilitate uncoating. Acidification weakens capsid interactions, leading to the release of protein VI, which disrupts the endosomal membrane, allowing escape into the cytoplasm. Partial disassembly exposes nuclear localization signals, enabling transport along microtubules toward the nuclear pore complex. Dynein motor proteins mediate this movement, ensuring efficient genome delivery to the nucleus.

Early Gene Expression

Once inside the nucleus, adenoviral DNA remains episomal, avoiding integration into the host genome. The virus relies on host RNA polymerase II to initiate early gene expression, regulated by the viral E1A protein. E1A activates transcription by interacting with chromatin modifiers and disrupting repressive histone marks. It also binds cellular factors such as p300/CBP and the retinoblastoma (Rb) family, releasing E2F transcription factors that drive early viral gene expression. By overriding cell cycle checkpoints, E1A ensures infected cells enter S-phase, favoring viral replication.

Early genes are expressed in a coordinated manner, each serving a distinct role. E1B proteins counteract host defenses, particularly E1B-55K, which binds p53 to prevent apoptosis. E2 gene products—DNA polymerase, preterminal protein, and single-stranded DNA-binding protein—prepare for genome replication. E3 proteins modulate intracellular trafficking, while E4 proteins aid in mRNA processing and nuclear export.

Alternative splicing enhances gene output from the compact genome. The adenoviral major late promoter (MLP) remains inactive during this stage to prioritize early functions. Viral non-coding RNAs, including VA-RNAs, accumulate to interfere with host RNA interference pathways, maintaining effective viral gene expression.

DNA Replication

Once early gene expression establishes a favorable environment, adenovirus replication begins within the nucleus. Unlike host chromosomal replication, which uses RNA primers, adenoviruses employ a terminal protein (TP) at the 5’ ends of their genome to initiate DNA synthesis. Viral DNA polymerase and preterminal protein (pTP) form a replication initiation complex, with pTP serving as a primer for polymerase extension. This mechanism eliminates the need for Okazaki fragments, ensuring continuous, strand-displacement replication.

As polymerase progresses, the displaced single-stranded DNA is rapidly coated by adenoviral single-stranded DNA-binding protein (DBP), preventing secondary structures and degradation. DBP stabilizes the unwound strand and enhances polymerase efficiency. Replication proceeds bidirectionally, generating full-length progeny genomes that remain associated with DBP until packaging. Host factors such as topoisomerases and nuclear scaffold proteins prevent excessive supercoiling and facilitate genome organization.

Late Gene Expression And Assembly

As DNA replication progresses, gene expression shifts to favor late-phase transcripts necessary for virion assembly. This transition is driven by the major late promoter (MLP), which becomes highly active once sufficient genome copies accumulate. The MLP generates a primary transcript that undergoes extensive alternative splicing and differential polyadenylation, yielding multiple late mRNAs encoding structural proteins. These include hexon, penton, and fiber proteins, as well as scaffolding components essential for virion assembly. VA-RNAs counteract host RNA interference pathways and enhance translation by inhibiting PKR, a kinase that suppresses protein synthesis in response to viral infection.

Capsid proteins undergo a regulated assembly process within the nucleus. Hexon trimers self-associate and are transported into the nucleus, where they integrate with penton bases and fiber proteins to form the complete icosahedral shell. Viral DNA is packaged into pre-assembled capsids through a mechanism involving the adenoviral packaging signal, which directs genome incorporation. ATP-driven molecular motors actively translocate DNA into the capsid, ensuring precise genome encapsidation. As packaging nears completion, viral proteases cleave precursor proteins, finalizing capsid maturation. Once assembled, virions accumulate in the nucleus, awaiting release.

Virion Release

Adenoviruses exit the host cell through lysis rather than budding. This process is mediated by the adenoviral death protein (ADP), which increases membrane permeability and promotes apoptosis. ADP disrupts the nuclear envelope, leading to fragmentation and cytoplasmic leakage. Host caspases further dismantle cellular components, weakening intercellular junctions and promoting viral spread.

As the host cell disintegrates, mature virions are passively released into surrounding tissues and fluids. This lytic strategy maximizes dissemination but also exposes the virus to immune defenses. The extent of cellular destruction varies by serotype, with some inducing rapid cytopathic effects while others maintain prolonged infection before lysis. Once outside the host, virions remain stable, allowing them to persist in diverse environments until they encounter a new host.

Interactions With The Immune System

Adenoviruses engage in a complex interplay with the immune system, using multiple strategies to evade detection while also triggering strong immune responses. Host cells recognize viral components through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and cytosolic DNA sensors, activating interferon-stimulated genes (ISGs) that restrict replication and recruit immune cells. Despite this response, adenoviruses encode proteins that counteract innate defenses. E1A and E1B interfere with apoptosis and interferon signaling, while E3 gene products modulate intracellular trafficking to avoid immune recognition.

Adenoviruses also elicit strong adaptive immunity, making them useful as vaccine vectors. Infected cells present viral antigens via major histocompatibility complex (MHC) molecules, stimulating CD8+ T cells. However, the virus counteracts this by expressing E3-19K, which retains MHC class I molecules in the endoplasmic reticulum, reducing antigen presentation. Despite these evasion tactics, the immune system eventually mounts a neutralizing antibody response, primarily targeting hexon and fiber proteins. This humoral immunity provides long-term protection but also poses challenges for adenovirus-based therapeutics, as preexisting antibodies can limit the efficacy of adenoviral vectors in gene therapy and vaccines.