Exploring the Replication Mechanisms of DNA and RNA Viruses

Abstract

This chapter provides an overview of the viral replication cycle, focusing on DNA and RNA viruses. The chapter begins with an introduction to viruses, their classification into DNA and RNA viruses, and their general characteristics. The process of DNA replication by viruses is then explained, including the different stages involved in this process and the specific mechanisms used by different types of DNA viruses. Similarly, the replication process of RNA viruses is discussed, with a focus on the various strategies employed by these viruses to replicate their genome. The purpose of this study is to investigate the intricate mechanisms involved in viral replication, which is essential for understanding the biology of viruses and developing strategies to control viral infections.

1. Introduction

Viruses are enigmatic entities, challenging the boundary between life and nonlife. Comprising short DNA or RNA sequences encased in protein shells, they remained invisible until the electron microscope’s invention. Bacteriophages, the first visualized viruses, possessed head- and tail-like structures, allowing only their nucleic acid to penetrate bacterial cells. Animal viruses displayed spherical or rod-shaped forms, interacting with cellular receptors [1].

Originally, size and filterability determined viruses classification, but recent advances in nucleic acid homology-based methods have reshaped the field [1, 2].

The infectious particles of viruses, or virions, harbor RNA or DNA genomes, which can be single- or double-stranded, linear, circular, or segmented. Some form nucleocapsid complexes with cellular histones or viral proteins, while others have capsids surrounding their nucleic acid [3]. Capsids, composed of repeating protein structures, provide vital protection, safeguarding the fragile nucleic acid from enzymatic degradation, physical stress, and radiation [4].

The Figure 1, showed a summary of the viruses discussed in this chapter.

Figure 1.

This visual summary provides key information about the viruses discussed in this chapter, highlighting genome type, virus family, and the primary host target cells.

Viruses continue to exert substantial evolutionary pressures, shaping cells, human health, and the global ecosystem [5].

DNA and RNA viruses employ a fascinating array of strategies to exploit host chaperones during their replication cycle. These chaperones, vital for normal cellular functions, are co-opted by viruses for various entry steps, such as cell attachment, endocytosis, unfolding, disassembly, and membrane penetration and offering potential targets for antiviral therapies, while others exploit their unexpected, noncanonical roles [6, 7].

A deep understanding of viruses serves as the foundation for creating, advancing, and using measures to prevent and treat diseases. The impressive progress in using viral vectors-based vaccines against COVID-19, vaccines clearly demonstrates how viral tools can quickly respond to health emergencies [8].

The viral vaccines show great potential role in addressing established infectious diseases and chronic noncommunicable diseases due to their safety, specificity, and effectiveness [7, 8].

The virus replication cycle, a complex process unfolding in several stages, plays a pivotal role in comprehending viral infections and formulating antiviral strategies. This cycle initiates with attachment, as viruses specifically bind to host cell receptors. Subsequently, penetration allows viruses to enter cells, exploiting endocytosis or fusion mechanisms. Uncoating involves capsid breakdown, releasing genetic material. Replication encompasses the core of the cycle, with different virus classes replicating and transcribing their genetic material. Assembly stage and the creation of new virions, and maturation finalizes infectious particles. Release concludes the cycle, with budding or cell lysis. Understanding this cycle is critical for combating viral infections [9, 10].

In conclusion, delving into the intricacies of the virus replication cycle is vital for grasping their infectious tactics, formulating effective antiviral interventions, pioneering technologies rooted in the virus life cycle, and expanding our comprehension of these extraordinary entities to safeguard humanity from the perils of viral infections [7, 11].

2. RNA viruses

RNA viruses, known for their swift adaptability and genomic variability, provide a fertile ground for scientific exploration. Their propensity for rapid mutation challenges conventional understanding of disease dynamics. In the upcoming section, we delve into the intricate world of RNA viruses’ replication strategies, focusing on specific examples that shed light on the mechanisms governing their proliferation. Table 1 provides a detailed examination of the general features and comparative analysis of various RNA viruses.

	HAV	HCV	Influenza A virus	Coronaviruses	HIV	Rubella virus
Virus family, genus	Picornaviridae, Hepatovirus	Flaviviridae, Hepacivirus	Orthomyxoviridae,; Alphainfluenzavirus	Coronaviridae; Alphacoronavirus/ Betacoronavirus/ Gammacoronaviru/ Deltacoronavirus	Retroviridae, Lentivirus	Matonaviridae, Rubivirus
Genome type	Single-stranded, positive-sense linear ssRNA	Positive-sense linear ssRNA	Eight single-stranded RNA segments	Single-stranded positive-sense RNA	Two identical single-stranded RNA	Single-stranded positive-sense RNA
Approx. genome length (nt)	7500	9600	13,500	26,000 to 32,000	9200–9600	9800
Envelope	No	Yes	Yes	Yes	Yes	Yes
Transmission	Mainly fecal–oral	Boold, Vertical, Parenteral, Sexual	Droplet	Droplet	Blood, Sexual, Injection drug use	Respiratory aerosols
Incubation period	28 days (range: 15–50 days)	7 weeks (range, 4–20 weeks)	2 days (range: 1–4 days)	5–6 days (range: 2–14 days)	3 weeks (range: 1–6 weeks)	14 days (range: 12–23 days)
Treatment	Supportive care,; Hydration	Supportive care, antiviral therapy for chronic liver disease	Supportive, antiviral medication to fight the infection	Antiviral treatments including oral antivirals or an IV antiviral	Antiretroviral therapy	Supportive
Prevention	Vaccination, immune globulin, personal hygiene, drinking safe water	Screening, avoiding direct exposure to blood or blood products	Vaccination, personal hygiene, avoiding close contact with patients	Vaccination, avoiding close contact with patients, wearing mask, personal hygiene	Avoiding risky sexual behaviors, safe sex practices, avoiding drug injection	Vaccination, herd immunity

Table 1.

A detailed examination of the general features and comparative analysis of RNA viruses discussed in this chapter.

2.1 Hepatitis a virus (HAV)

Hepatitis A virus (HAV) replication cycle is a complex process that consists of several stages. The cellular entry of HAV involves quasi-enveloped virus interactions with cell receptors, primarily mucin domain-containing protein 1 (TIM1) and T-cell immunoglobulin. Although the exact cellular types and primary replication sites remain uncertain, spread within the host mainly involves quasi-enveloped HAV [12, 13].

Upon entry, quasi-enveloped eHAV is taken up through mechanisms similar to those of exosomes [14].

Viral protein translation commences, driven by the internal ribosome entry site (IRES) within the viral genome. The translation produces both structural and nonstructural proteins essential for RNA replication. The replication process involves minus-strand RNA synthesis, guided by an uridylated VPg primer. Positive-strand synthesis follows, utilizing the same VPg-pUpU primer [12, 15].

The synthesis of new genomes and subsequent uncoating of HAV proceed within membranous replication factories, likely derived from the endoplasmic reticulum. Host factors contribute to the establishment and maintenance of these replication complexes.

After viral RNA synthesis, the assembly of HAV capsids and packaging of RNA take place within infected cells. The capsids and dsRNA colocalize, suggesting maturation occurs near sites of RNA replication. Viral egress is predominantly nonlytic, involving interactivity with the components of the ESCRT system. The viral capsids are recruited to endosomes, forming multivesicular bodies (MVBs) that convey capsids to the plasma membrane for release [16].

The detailed mechanisms underlying these processes are still being explored. HAV’s ability to quasi-envelope itself, along with its distinct replication strategies, plays a decisive role in its survival and transmission, both within the host and in the external environment [12].

In the context of hepatitis A, despite the effectiveness of vaccines, the need for antiviral treatments is a significant concern. Host-targeting agents (HTAs), such as interferon types I and III and the human La protein, along with small interfering RNAs and Janus kinase inhibitors, have shown promise in inhibiting HAV replication. Additionally, zinc compounds and heme oxygenase-1 exhibit potential for blocking viral replication. Established antiviral drugs like ribavirin, amantadine, and sofosbuvir have demonstrated activity against HAV. Direct-acting antivirals focus on HAV IRES, 3C protease, and 3D polymerase. While entry pathway inhibition is explored, further research is needed to identify HAV cell surface receptors [17].

2.2 Hepatitis C virus (HCV)

Hepatitis C virus (HCV), a positive-strand RNA virus within Flaviviridae, orchestrates a complex replication cycle encompassing key stages. HCV is a significant global pathogen, infecting approximately 350 million people worldwide. It often leads to chronic infections in 60–80% of affected individuals, with some developing severe liver damage, cirrhosis, and a high risk of hepatocellular carcinoma (HCC) [18].

HCV’s pathogenesis is influenced by the host’s immune response and its direct impact on liver cell proliferation and viability, which has implications for liver damage and carcinogenesis.

Taxonomically, HCV belongs to the Hepacivirus genus in the Flaviviridae family. Its genome is a positive, single-stranded RNA molecule with an open reading frame encoding structural and nonstructural proteins, processed with the help of host and viral-encoded enzymes [19].

The initial step to HCV replication cycle involves HCV entering hepatocytes, its primary host cells. This intricate process hinges on interactions between the virus’s envelope glycoproteins (E1-E2) and glycosaminoglycans (GAGs), facilitating initial binding. Furthermore, there have been suggestions of connections between low-density lipoproteins (LDL) and the LDL receptor (LDLr) that warrant investigation. The subsequent engagement of scavenger receptor BI (SR-BI), Occludin (OCLN), CD81 tetraspanin, and Claudin-1 (CLDN-1) results in clathrin-dependent HCV uptake, complemented by high-density lipoprotein’s enhancement of entry kinetics. As the endosomal pH drops, glycoprotein conformational changes trigger viral-endosomal membrane fusion, culminating in uncoating [20, 21].

When it enters, the virus delivers its RNA into the cytoplasm, where it undergoes translation via an internal ribosome entry site (IRES) located at the rough endoplasmic reticulum (ER). This translation yields a polypeptide that undergoes cleavage, yielding 10 distinct products, including core protein derivatives. Every viral protein, whether through direct interaction or indirectly via NS4A, forms associations with ER-derived membranes. The membranous replication vesicles (RVs), forming a structure known as the membranous web, are induced primarily by NS4B, potentially in conjunction with NS5A. Within this web, viral RNA amplification transpires, guided by NS5B RNA-dependent RNA polymerase (RdRp) and various host factors. This process includes the creation of a negative-strand RNA template, which is subsequently utilized for both replication and translation purposes [21, 22].

Notably, the assembly phase involves core protein accumulation on lipid droplets (LDs), pivotal for viral particle formation. Viral RNA is delivered to these LDs by either the replicase or NS5A. Subsequent interactions between core protein and RNA may trigger nucleocapsid formation, which may bud into the endoplasmic reticulum (ER) lumen, closely tied to very low-density lipoprotein (VLDL) synthesis. These complex processes are targeted by antiviral drugs that address various stages of replication, encompassing enzymes like NS3 protease, NS5B RdRp, and NS5A [21, 23].

In the hepatitis C virus (HCV) replication cycle, several critical steps are targeted by direct-acting antiviral drugs (DAAs). These drugs are essential for disrupting the virus’s ability to replicate and proliferate within the host’s cells. They focus on key viral proteins, including protease inhibitors (e.g., glecaprevir) that inhibit NS3 protease, crucial for processing the viral polyprotein. This disruption hampers the maturation of viral proteins required for replication. RDdRp inhibitors (e.g., sofosbuvir) target the NS5B RNA-dependent RNA polymerase, responsible for replicating the viral RNA genome, effectively halting replication. Moreover, NS5A inhibitors (e.g., velpatasvir) address the multifunctional NS5A protein involved in various aspects of RNA replication and viral particle assembly. Antiviral drugs that address these specific enzymes interfere with critical steps in the HCV replication cycle, ultimately preventing the virus from multiplying. These DAAs offer high efficacy, minimal side effects, and short, oral treatments, making them a cornerstone of HCV treatment strategies for both acute and chronic infections. Glecaprevir-pibrentasvir (8 weeks) and sofosbuvir-velpatasvir (12 weeks) are recommended for chronic HCV, while sofosbuvir-velpatasvir-voxilaprevir (12 weeks) is suggested in case of virological failure. [21, 23, 24]

2.3 Influenza a virus

Influenza A virus, a member of the Orthomyxoviridae family, is the cause of regular flu outbreaks and global pandemics. One interesting thing about it is how its genetic information is divided into eight separate parts, like different chapters in a book. Each part contains instructions for making various proteins that the virus needs to reproduce and spread.

It is essential to know that while we have learned a lot about the virus’s proteins, there is still much we do not understand. New research keeps showing us how complex the virus is. This ongoing exploration is crucial to learn more about the virus and develop ways to fight it.

Influenza A comes in different types, depending on the outer parts of the virus, called hemagglutinin (HA) and neuraminidase (NA). This virus can quickly change its genetic makeup, making it tough for health experts. That is why we need to make new vaccines each year that match the latest versions of the flu virus to keep people safe [25, 26].

Within the virion, vRNA segments assemble into ribonucleoprotein complexes (vRNPs). These vRNPs consist of the highly conserved 5′ and 3′ termini of vRNA, which interact through base-pairing to form a partially double-stranded structure. This structure is then encapsulated by a trimeric RNA-dependent RNA polymerase complex, while the remaining portion of the vRNA associates with multiple copies of oligomeric nucleoprotein (NP). A vRNA segment containing the bound RNA polymerase and NP represents the virus’s minimal transcriptional and replicative machinery [25, 27].

The replication cycle commences with the initial attachment of the infecting virion to cell surface receptors containing sialic acid, after which the virion is internalized via endocytosis. The subsequent fusion event between viral and endosomal membranes facilitates the release of vRNPs into the cytoplasm, where they are then transported into the cell nucleus. Within the nucleus, the viral RNA polymerase orchestrates the transcription of vRNA segments into mRNAs, which undergo 5′ capping and 3′ polyadenylation. These mature mRNAs are subsequently exported to the cytoplasm, where they undergo translation via cellular machinery. Concurrently, the viral RNA polymerase initiates vRNA replication, generating complementary RNA (cRNA) as a template for the production of additional vRNA. Both cRNA and vRNA are then assembled along with newly synthesized viral polymerase and NP, giving rise to cRNPs and vRNPs, respectively. After their exit from the nucleus, progeny vRNPs traverse the cytoplasm through a mechanism dependent on Rab11 and microtubules, ultimately reaching the cell membrane. It is at the cell membrane that the assembly of progeny virions takes place, culminating in their release via budding [28].

Importantly, it should be highlighted that the precise packaging signals situated within both the 5′ and 3′ termini of individual genome segments, encompassing noncoding as well as coding regions, play a pivotal role in enabling the incorporation of all eight genome segments into virions. Nevertheless, the exact characteristics of these packaging signals and the mechanisms underlying the selection of segments for packaging continue to pose unresolved questions.

While the replication cycle of influenza A virus primarily transpires in the nucleus, several intriguing features set it apart from other RNA viruses. The genome’s negative polarity necessitates transcription into functional mRNA before replication can commence. Additionally, independent replication of the eight RNA genes in the segmented and single-stranded genome poses challenges in progeny virus assembly. Remarkably, replication involves transcription of host cell DNA without requiring new DNA synthesis, a process that remains one of animal virology’s unsolved mysteries [25].

Further investigations have revealed a dependence on host cell functions for influenza virus replication. During early infection, host cell RNA synthesis is transiently stimulated, occurring in the nucleoplasm and involving a-amanitin-sensitive RNA polymerase II activity. This enzyme is crucial for virus replication, as influenza can replicate in mutant cells with altered, drug-resistant RNA polymerase II. High doses of a-amanitin or actinomycin D can inhibit influenza replication by affecting the availability of RNA polymerase II enzyme subunits in the virus transcription complex [29].

The synthesis of virion RNA is a dynamic process, with results varying depending on experimental approaches. Studies have shown that vRNA synthesis begins shortly after infection and peaks at different times, highlighting the complex kinetics of RNA synthesis during influenza infection. Additionally, non-polyadenylated cRNA is proposed to serve as the template for vRNA synthesis. This type of RNA is detectable soon after infection, accumulating rapidly in the cytoplasm and more slowly in the nucleus. The increase in cRNA precedes that in vRNA, supporting the hypothesis that non-polyadenylated cRNA acts as a template [25, 28].

Antiviral drugs for influenza are primarily neuraminidase inhibitors (NAIs) and cap-dependent endonuclease inhibitors (CENIs). Common NAIs include zanamivir, oseltamivir, peramivir, and laninamivir, while a unique CENI, baloxavir marboxil, is available. Baloxavir stands out for its single-dose therapy, an advantage over oseltamivir’s longer regimen. These drugs have a favorable safety profile, with common side effects like headaches and gastrointestinal issues. Administering them within 12 hours of symptom onset enhances their effectiveness, potentially shortening treatment periods. While not achieving optimal therapeutic benefits, these drugs reduce complications and work well in prophylaxis, with vaccination remaining the most effective flu prevention method [30].

In summary, influenza A virus follows a unique replication cycle that starts with the virion binding to cell receptors, leading to the release of vRNPs into the nucleus, where transcription and replication occur. The nature of packaging signals and the mechanism behind segment selection for virion assembly remain unknown. Moreover, the virus’s dependence on host cell functions and the intricacies of RNA synthesis kinetics during infection pose intriguing research challenges.

2.4 Coronaviruses (CoVs)

Coronaviruses (CoVs) are members of the Coronaviridae family, comprising four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Their genome is a single-stranded positive-sense RNA (+ssRNA) of about 30 kb with a 5′-cap and 3′-poly-A tail. Initially, this RNA translates polyprotein 1a/1ab, which forms the replication-transcription complex (RTC) within double-membrane vesicles (DMVs). Afterward, subgenomic RNAs (sgRNAs) are produced via discontinuous transcription by RTC. These sgRNAs possess common 5′- and 3′-end sequences and serve as templates for structural and accessory protein synthesis [31, 32].

CoV genomes contain at least 6 open reading frames (ORFs), encoding nonstructural proteins and structural proteins like spike, membrane, envelope, and nucleocapsid. CoVs also encode specific structural and accessory proteins. Notably, CoV genomes are large for RNA viruses, almost twice the size of the second-largest RNA viruses. This large size is related to features of the CoV RTC, including the unique 3′-5′ exoribonuclease, which functions as part of the RTC’s proofreading mechanism [31, 33].

The replication cycle of CoV commences with the binding of the virion to the host cell, primarily through interactions involving the spike protein and its receptor. S protein mediates binding to host receptors and undergoes conformational changes leading to virion-cell membrane fusion. Different CoVs use specific receptors, such as ACE2 for SARS-CoV, DPP4 for MERS-CoV, and various receptors for other CoVs. After receptor binding, proteolytic cleavage of the S protein enables the virus to enter the host cell cytosol [34].

The initiation of viral RNA synthesis occurs subsequent to the translation and formation of the viral replicase complexes. This sequential event leads to the generation of both genomic and subgenomic RNAs, with the latter functioning as templates for the expression of structural and accessory genes. CoVs exhibit unique nested RNAs due to their distinctive replication process, involving negative-strand intermediates.

CoV replication is controlled by several cis-acting sequences within the genome’s 5′ and 3′ untranslated regions. A particularly intriguing aspect of CoV replication is how the leader and body transcription regulatory sequence (TRS) segments fuse during subgenomic RNA production. This involves discontinuous extension of negative-strand RNA, guided by the complementarity of TRS-B to the leader TRS (TRS-L). Details regarding the transition from discontinuous to continuous transcription are still under investigation [33, 35].

CoVs are recognized for their capacity to engage in recombination, encompassing both homologous and nonhomologous, which contributes to viral evolution. This recombination is closely tied to the RNA strand-switching capacity of the viral RdRp. Recombination has a crucial role in viral evolution and is employed in reverse genetics tools for engineering viral recombinants [34].

Several antiviral drugs show potential against SARS-CoV-2. They employ diverse mechanisms, including fusion inhibitors like EK1C4 and arbidol, NAs such as remdesivir and ribavirin, and protease inhibitors like lopinavir, ritonavir, darunavir, and disulfiram. These drugs offer various strategies to disrupt viral replication and reduce infection severity. Clinical trials are assessing their effectiveness in COVID-19 treatment [36].

In summary, the replication cycle of coronaviruses involves attachment to host cells through spike protein-receptor interactions, followed by RNA synthesis, subgenomic RNA production, and protein translation. CoVs are known for their large genomes, recombination abilities, and distinctive replication processes involving nested RNAs [35].

2.5 Human immunodeficiency virus (HIV)

Human immunodeficiency virus (HIV) belongs to the Lentivirus genus within the Retroviridae family, under the Orthoretrovirinae subfamily. It is further divided into types 1 and 2 (HIV-1, HIV-2) based on genetic and antigenic differences. Lentiviruses, which include simian immunodeficiency viruses (SIV) from nonhuman primates, share genetic similarities [37, 38].

Epidemiological and phylogenetic analyses suggest HIV entered the human population between 1920 and 1940. HIV-1 originated from Central African chimpanzee immunodeficiency viruses (SIVcpz), while HIV-2 evolved from West African sooty mangabey immunodeficiency viruses (SIVsm).

HIV’s genetic material consists of two identical single-stranded RNA molecules enclosed within the virus particle’s core. During infection, the viral RNA is reverse-transcribed into proviral DNA, which integrates into the human genome [38].

The HIV replication cycle is a complex process involving several key stages. The process begins with the binding of the viral envelope glycoprotein gp120 to the CD4 receptor on the host cell’s surface. The interaction induces a structural shift in gp120, allowing it to attach to chemokine receptors like CXCR4 or CCR5. This double binding allows for a stable attachment of the virus to the host cell. Subsequently, the viral and cellular membranes fuse together [30].

After membrane fusion, the virus core is released into the cytoplasm of the target cell, marking the uncoating step. Upon entering the cell, viral RNA undergoes a process of reverse transcription, mediated by the enzyme reverse transcriptase. This process converts the viral RNA into a double-stranded DNA copy.

The enzyme integrase integrates the newly synthesized viral DNA into the genome of the host cell. This integrated form is called the provirus and remains within the host cell for its lifetime [39].

Transcription of the proviral DNA occurs, leading to the synthesis of various viral proteins, including regulatory proteins like Tat and Rev. and structural proteins like Gag, Pol, and Env. These proteins are essential for the assembly of new virions.

The assembly of new virions involves the formation of the virus capsid and envelope. The HIV-1 protease cleaves precursor molecules into their final forms, allowing for the generation of infectious viral particles. These new virions bud from the host cell’s membrane, acquiring a lipid bilayer containing viral glycoproteins [40].

Throughout the replication cycle, various host cell determinants, such as chemokine receptors and the activation state of the target cell, play crucial roles in determining viral tropism. HIV-1 can use different coreceptors, including CXCR4 and CCR5, and strains that can bind to both are termed dual tropic or X4R5 viruses [38, 40].

In the past decade, a range of reverse transcriptase inhibitors have been approved to combat HIV-1. These drugs are the result of multidisciplinary collaboration, with crystallography guiding their development. Clinical trials have shown the effectiveness of these regimens in reducing HIV infections. Standard treatment often combines two NRTIs with other drugs but may lead to adverse events.

Tenofovir alafenamide is replacing Tenofovir disoproxil fumarate. Non-nucleoside reverse transcriptase inhibitors are giving way to integrase inhibitors in initial regimens.

HIV strains evolve rapidly, leading to drug resistance. Novel strategies are explored to address this issue. Some RT inhibitors can be repurposed for other diseases, like HBV infections and diabetes. Future developments include more potent inhibitors, innovative applications, and extended-release delivery devices. These drugs have significantly impacted HIV treatment, and their evolution continues to adapt to the changing virus landscape [41].

The replication cycle involves intricate steps, including reverse transcription, integration, and the regulation of viral gene expression. After integration into the host cell’s genome, the provirus can enter a dormant state in certain cell types, necessitating activation to enable viral gene expression.

2.6 Rubella virus (RuV)

Rubella virus (RuV) is the causative agent of rubella, characterized by a mild rash. Structurally, RuV belongs to the Rubivirus genus within the enveloped Togaviridae family. It carries two glycoproteins, E1 and E2, essential for host cell entry. Its genome is a non-segmented single-stranded positive-sense RNA.

Taxonomically, RuV is categorized in the Togaviridae family and Rubivirus genus and is associated with congenital rubella syndrome (CRS) in pregnant women. To mitigate this risk, global rubella vaccination programs have been established, emphasizing the virus’s importance in public health [42, 43].

RuV likely utilizes a widespread host cell receptor, possibly involving membrane phospholipids and glycolipids, for attachment. The exact mechanisms involved in entry remain elusive, but evidence suggests that RuV may employ an endocytic pathway. Under lower pH conditions within endosomes, structural shifts in viral glycoproteins promote fusion with the endosomal membrane, facilitating viral entry [44, 45].

Following entry, the capsid protein of RuV undergoes conformational changes, potentially within endosomes at lower pH levels, allowing it to uncoat. This process enables the viral genomic RNA to be released into the cytoplasm of the host cell.

RuV replication is characterized by a protracted cycle featuring an extended viral latent period of 8 to 12 hours. During this phase, four unique viral RNA species are produced: genomic RNA, subgenomic RNA, replicative intermediates, and replicative forms. These RNA molecules serve as templates for synthesizing viral nonstructural proteins (NSPs) and structural proteins (SPs). The newly formed genomic RNA associates with capsid proteins to form nucleocapsids [46].

As the replication progresses, newly synthesized viral structural proteins assemble into virions, with only positive-strand genomic RNA being packaged into these new virions. Peak virus production typically occurs between 36- and 48-hour postinfection. Interestingly, RuV does not infect all host cells simultaneously, and the proportion of infected cells can vary based on cell type. Nevertheless, with time, the entire cell culture eventually becomes infected [46, 47].

Notably, RuV capsid protein (CP) plays a multifaceted role in the virus’s life cycle. While contributing to nucleocapsid formation during viral assembly, it also enhances viral genome replication in the early stages of infection. Intriguingly, experiments have demonstrated that exogenous RuV CP from input virus particles can enhance genome replication [47, 48].

While the exact mechanisms governing RuV uncoating and nucleocapsid disassembly are not fully elucidated, there is a belief that phosphorylation of RuV CP within virions is pivotal in aiding the release of the viral genome from nucleocapsids during disassembly. This process may differ substantially from the uncoating mechanisms observed in other viruses, such as alphaviruses [48].

Rubella lacks approved antiviral drugs and is challenging to treat due to the lack of a small animal model. Nitazoxanide showed promise in reducing rubella antigen, while nanchangmycin and various host-targeted drugs displayed antiviral activity. Nucleoside analogs, NM107 and AT-527, originally developed for HCV, exhibited potential for repurposing against rubella. Combinations of nucleoside analogs with nucleoside biosynthesis inhibitors enhanced antiviral effects. These findings suggest repurposing nucleoside analogs as a viable approach to treating rubella. Further research will explore the mechanisms underlying these antiviral actions [49].

In summary, RuV replication is a complex and dynamic process that encompasses attachment, entry, uncoating, replication, assembly, and release. Further research holds the potential to unveil the specific mechanisms involved in RuV replication and uncoating, particularly the contribution of CP to the enhancement of genome replication.

3. DNA viruses

DNA viruses, a fascinating group of microorganisms, wield the power of genetic material to commandeer host cells and replicate. Unlike RNA viruses, they store their genetic information as DNA, which is intricately intertwined with the cellular machinery. These viruses span diverse families and possess distinct strategies for infection, replication, and pathogenesis. In this part, we delve into the captivating world of DNA viruses, briefly examining the replication cycles of select members, shedding light on the fascinating processes that underpin viral replication. Table 2 breaks down the key features and comparisons of the DNA viruses we will discuss in this chapter.

	HBV	Adenovirus	Papillomaviruses	Poxviridae	Herpes simplex viruses
Virus family, genus	Hepadnaviridae,; Orthohepadnavirus	Adenoviridae, Atadenovirus	Papillomaviridae, Alphapapillomavirus/Betapapillomavirus/Gammapapillomavirus/Mupapillomavirus/Nupapillomavirus	Poxviridae, Orthopoxvirus	Herpesviridae, Simplexvirus
Genome type	Double-stranded circular DNA	Double-stranded linear dsDNA	Double-stranded circular DNA	Double-stranded linear DNA	Double-stranded linear DNA
Approx. genome length (nt)	3200	26,000–45,000	8000	135,000–360,000	152,000
Envelope	Yes	No	No	Yes	Yes
Transmission	Blood, Vertical, Sexual, Parenteral	Droplets	Sexual	Introduction of virus into skin abrasions, directly or indirectly from a contaminated environment	Contact with the virus in sores, saliva, or surfaces in or around the mouth
Incubation period	90 days (range 60–150 days)	Respiratory infection 2–14 days, Gastroenteritis 3–10 days	2–3 month (range 1–20 months)	10–14 days (range 7–19 days)	4 days (range 2–20 days)
Treatment	Antiviral therapy, Interferon therapy	Supportive care, Eye care for adenovirus conjunctivitis	Topical Treatments, Electrosurgery, Laser Therapy, Colposcopy, and Biopsy	Supportive care, Antiviral therapy	Antiviral therapy, Suppressive antiviral therapy
Prevention	Vaccination, Safe sex practices, Avoiding Sharing Needles and Personal Items that may come into contact with blood	Personal hygiene, Avoiding close contact with patients	Vaccination, Safe sex practices and limiting sexual partners, Routine Pap smears and cervical cancer screening	Vaccination, Isolation and quarantine (to prevent the spread of the virus to others), Personal hygiene	Safe sex practices

Table 2.

A detailed examination of the general features and comparative analysis of DNA viruses discussed in this chapter.

3.1 Hepatitis B virus (HBV)

Hepatitis B virus (HBV) is the culprit behind hepatitis B, a liver infection. HBV is classified under the Orthohepadnavirus genus within the Hepadnaviridae family, with human HBV causing the majority of cases, featuring a partially double-stranded DNA genome encased within a protein core. The viral envelope, derived from host cell membranes, contains surface proteins (HBsAg) vital for entering liver cells.

Hepatitis B is a major public health concern due to its potential for chronicity and liver disease, addressed through vaccination programs [50, 51].

As a result of the lack of a viable cell culture system for propagating HBV, much of our knowledge has been acquired through genetic approaches using DNA transfection into cells. This technique permits just one cycle of virus generation, obscuring the early stages of infection. The virus probably attaches to hepatocytes via a portion of the preS region found on the large envelope proteins, even though the specific cellular receptor and the process of viral genome uncoating remain unclear [52].

Transcription is a critical stage in the HBV replication cycle. Genomic and subgenomic RNAs are transcribed from internal promoters, giving rise to a variety of transcripts that serve as templates for different viral proteins. These transcripts are capped, unspliced, and polyadenylated, representing a unique feature of HBV [52, 53].

In the cytoplasm, core protein, RNA pre-genome, and P protein accumulate. These three elements come together to form core particles, which serve as the apparatus for replication. It is worth mentioning that the P protein assumes a crucial function in RNA encapsidation and exhibits a preference for its own mRNA during this process.

Reverse transcription is a fundamental process that transforms the encapsidated RNA pre-genome into partially double-stranded circular DNA (cccDNA). This intricate process begins at the 3′ end of the RNA template and proceeds toward the 5′ end. Circularization of the DNA involves complex priming and extension processes [53].

The outcome of this process is the formation of cccDNA, a unique molecule organized in a chromatin-like structure. This cccDNA serves as the template for transcription, where both host transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) and hepatocyte nuclear factors (HNF), along with viral proteins, are indispensable for governing the transcription process [54].

Following translation, complex formation and reverse transcription occur. The pre-genomic RNA (pgRNA), core protein, and polymerase interact, initiating the key process of reverse transcription. This phase represents the maturation of nucleocapsids from RNA-containing to DNA-containing [53].

Finally, DNA-containing nucleocapsids have two potential paths. They have the potential to be transported back into the nucleus, thereby participating in the formation of extra cccDNA molecules. Alternatively, they can be enveloped, ultimately leading to the secretion of noninfectious subviral envelope particles (SVPs) [52, 53].

This detailed overview provides insight into the remarkable complexity of the HBV replication cycle, involving specific viral components, intricate processes, and interactions with host factors.

NUC therapy is the primary treatment for chronic hepatitis B (CHB) in most patients, with a growing trend toward finite NUC therapy. Predicting outcomes after NUC discontinuation remains challenging, but EOT qHBsAg <100 IU/mL is a promising predictor.

Monitoring post-NUC treatment is essential, especially for patients with liver decompensation. Assessing whether retreatment is needed is crucial. Future CHB treatments aim to target various stages of the hepatitis B virus (HBV) life cycle and enhance the host’s immunity. These investigational drugs are in early development stages, with a focus on achieving higher HBsAg loss rates [55].

3.2 Adenovirus (HAdV)

The Adenovirus (HAdV) replication cycle is a highly efficient and complex process that involves various stages and an intricate interplay of viral and cellular components. At its core lies the HAdV genome, a linear double-stranded DNA of approximately 36 kb, featuring inverted terminal repetitions. A 55-kDa terminal protein (TP) is covalently linked to these termini. Remarkably, infected cells can produce about one million copies of viral DNA within a mere 40 hours [55].

The key to this replication process is the origins of replication, where DNA synthesis initiates. HAdV boasts two identical origins situated within the inverted terminal repeats. These origins comprise a minimal origin, consisting of the terminal 18 bp, and an auxiliary origin. The initiation of DNA synthesis is a multifaceted process orchestrated by various viral proteins, including pTP, DNA-binding protein (DBP), and AdV DNA polymerase (AdV Pol). Crucially, this initiation is greatly enhanced by the involvement of cellular transcription factors, Oct-1 and NFI [56, 57].

The formation of a preinitiation complex marks a pivotal phase. Here, complex interplay between viral and cellular proteins and the origins leads to structural changes within the origins. These changes are primarily induced by NFI and Oct-1, which result in extensive origin bending [56].

A distinctive feature of HAdV DNA replication is the initiation at an internal site, where replication begins at a specific location on the template. Subsequently, the formation of a pTP-trinucleotide intermediate, pTP-CAT, occurs. A notable aspect of this mechanism is the “jumping-back” phenomenon, where the pTP-CAT intermediate shifts back three bases and pairs with template residues 1–3 [58].

Another critical facet is primer usage, where a specific amino acid within pTP acts as a primer for the covalent attachment of dCMP by AdV Pol. This step ensures efficient polymerization and proofreading of the newly synthesized DNA.

As replication progresses, the preinitiation complex undergoes disassembly. The dissociation of NFI occurs early in the initiation process, while Oct-1 remains associated until the recognition site becomes single-stranded upon replication fork passage [56, 57].

Ultimately, the culmination of these intricate steps leads to the efficient elongation of the pTP-CAT intermediate by AdV Pol and DBP. This leads to the creation of a fresh duplex genome, accompanied by the displacement of the nontemplate strand. Interestingly, displaced nontemplate strands may anneal together, potentially serving as substrates for subsequent rounds of replication [56].

In a broader context, this mechanism of adenovirus DNA replication bears similarities to protein-primed DNA replication found in various bacteriophages. These bacteriophages, like HAdV, possess linear double-stranded DNA genomes and employ similar sliding-back or jumping-back mechanisms.

The viral replication cycle offers potential targets for antivirals. Some compounds, such as niclosamide, oxyclozanide, and rafoxanide, interfere with different stages of the HAdV life cycle. Epigenetic regulators play a crucial role in HAdV replication. Drugs like gemcitabine, chaetocin, lestaurtinib, HDAC inhibitors, GSK126, and GSK343 can disrupt HAdV replication by altering epigenetic modification [59].

3.3 Papillomaviruses (PVs)

Papillomaviruses (PVs) are known for their unique replication cycle, which capitalizes on the regenerative process within stratified epithelial tissues. Understanding this cycle is pivotal for unraveling PV infections and devising potential therapeutic strategies.

PV replication unfolds through a series of stages. Initially, upon infecting basal cells, PV genomes undergo rapid replication, generating a low copy number. This initial amplification is an important setup phase for further replication [60, 61].

In the subsequent phase, known as “establishment,” the viral genome must secure a stable extrachromosomal replicon status within the host nucleus. This step is vital for ensuring a persistent infection. It involves intricate interactions of the viral DNA with host chromatin, which not only determine its location but also prevent it from being silenced or restricted [60, 61].

As the infection progresses, the maintenance phase becomes essential. During this period, PV genomes maintain a constant copy number and skillfully partition into daughter cells during cell division. This efficient retention of viral DNA extrachromosomally is a cornerstone of long-term persistence [61].

In differentiated epithelial cells, the vegetative amplification stage takes center stage. Here, high copy numbers of viral genomes are generated, intended to be packaged as progeny virions. This continuous production of viral particles serves as a crucial strategy for PV to evade the immune system.

Several key factors come into play throughout these stages. The E1 and E2 proteins, for instance, play multifaceted roles. E1 is pivotal in establishing the viral genome as a nuclear plasmid upon infection and continues to support its long-term maintenance. E2, conversely, is a versatile player engaged in transcription activation, DNA replication initiation, and maintenance [62, 63].

The E6 and E7 oncoproteins are also important contributors. They create a cellular environment favorable to PV persistence and manage to bypass checkpoints that would normally impede the extended long-term presence of extrachromosomal DNAs [60, 64].

Interactions with host chromatin are fundamental throughout these processes. Various cellular proteins, including TopBP1, ChlR1, and Brd4, are implicated at different stages of PV maintenance, ensuring the viral genome’s stability [61, 65].

One interesting aspect is the mode of PV DNA replication during stable maintenance. It can vary, with some studies suggesting a once-per-cell-cycle pattern, while others propose a more random replication. The outcome may depend on the cell type and the expression level of the E1 protein [62].

MEK1/2 inhibitors are effective against papillomavirus (PV)-induced tumorigenesis, reducing early gene transcription and oncoprotein functions in PV. They work without T cells, benefiting immunocompromised individuals. Treating pathologically confirmed that the disease aligns with clinical relevance, showing that MEK inhibitors significantly reduce tumor growth by inhibiting cellular proliferation and early PV gene transcription. Targeting both MEK1 and MEK2 is vital for controlling PV disease [66].

In summary, papillomaviruses employ a sophisticated replication strategy, encompassing initial amplification, establishment, maintenance, and vegetative amplification stages. These stages are intricately orchestrated by viral proteins like E1, E2, E6, and E7 and involve complex interactions with host chromatin.

3.4 Poxviridae

The replication cycle of Poxviridae is a complex and intriguing process that unfolds within the host cell’s cytoplasm. Poxviruses exhibit a unique strategy, replicating exclusively in the cytoplasmic environment.

Upon infection, poxviruses enter the host cell and release their core into the cytoplasm. This core is transported along microtubules (MT) toward the microtubule-organizing center (MTOC). During this journey, vimentin undergoes rearrangement, chaperones are recruited, and mitochondria gather around the core. These events culminate in the formation of replication compartments [67].

Remarkably, early transcription of genes commences within the viral core itself, driven by the viral DNA-dependent RNA polymerase and packaged viral transcription factors. About 100 early mRNAs are produced and then transported into the cytoplasm, where they engage ribosomes for translation. Simultaneously, viral cores accumulate near the rough endoplasmic reticulum (rER). Early proteins facilitate the uncoating of the viral core and the release of viral DNA once the core reaches the ER [60, 68].

Genome uncoating depends on ubiquitin-mediated proteasomal degradation of capsid proteins that were previously ubiquitinated. Ubiquitin ligases are crucial for genome replication. The released genome associates with viral proteins responsible for DNA replication and organization of the replication compartment forerunner [67].

As DNA replication proceeds, intermediate and late mRNAs, along with ribosomes and translation factors, gather within enlarging DNA factories. These factories are intricately associated with the rER. The extensive repertoire of poxvirus-encoded proteins involved in transcription, RNA synthesis, and DNA replication is complemented by the recruitment of numerous cellular factors during infection [69].

Throughout the infection cycle, Poxviridae adeptly manipulate cellular processes. Cellular protein synthesis is downregulated, and cellular mRNA is degraded, promoting selective translation of viral mRNAs. Translation of viral mRNA primarily transpires within viral replication compartments, ensuring efficient viral gene expression.

In the late stage of infection, as the assembly of virions begins, replication sites are released from the rER. Crescent-shaped membranes interact with viral DNA, initiating the intricate process of virion assembly [67, 68].

Regarding DNA replication models, Poxviridae are believed to employ a self-priming mechanism, potentially resembling the rolling hairpin strand-displacement mechanism observed in parvoviruses. DNA hairpins located at the termini of the poxvirus genome imply a self-priming model. An unidentified nuclease creates a nick in proximity to the hairpin, supplying a 3′ OH end for deoxynucleotide addition. The complementary strands fold back, facilitating deoxynucleotide addition to the distal hairpin and around it, ultimately forming concatemers.

In conclusion, the Poxviridae replication cycle is an intricate and highly orchestrated process occurring exclusively within the cytoplasm. It involves a cascade of events, from core release to genome uncoating, DNA replication, and translation, all within the confines of specialized replication compartments [67, 68, 70].

Antiviral drugs and vaccines play a crucial role in managing infections caused by Orthopoxviruses, including mpox. Vaccination with live VACV vaccine ACAM2000 and MVA-based vaccine JYNNEOS has shown promising immune responses. However, these vaccines may have side effects, and further research is needed to optimize their usage. Antiviral drugs like tecovirimat target specific viral proteins to inhibit viral replication, while brincidofovir and cidofovir act on viral DNA synthesis, inhibiting replication. VIG is another drug showing potential against mpox. Combining vaccination, antiviral drugs, and public health strategies is crucial to control mpox and prevent its potential spread from endemic to pandemic status [71].

3.5 Herpes simplex viruses (HSVs)

The Herpes Simplex Virus (HSV) replication cycle is a highly intricate and tightly regulated process that occurs within host cells. HSV-1 and HSV-2 exhibit shared characteristics in their replication cycles.

The HSV genome is a large and structurally complex entity, comprising unique long and unique short regions flanked by inverted repeat sequences. Central to initiating viral DNA synthesis are the origins of replication, OriL and OriS. These origins, abundant in A/T sequences, play a vital role in the initiation of viral DNA replication. Key proteins such as UL9 and ICP8 play crucial roles in this initiation phase. They work together to alter the AT-rich origin spacer region, promoting the formation of DNA structures that mark the beginning of viral DNA replication [72, 73].

As HSV infection progresses, it triggers a significant reorganization of the host cell nucleus. This results in the creation of specialized domains within the nucleus, referred to as replication compartments (RCs). RCs serve as key environments for various viral processes, such as gene expression, DNA synthesis, and the formation of viral nucleocapsids. Notably, essential viral proteins like ICP0, ICP4, and ICP27, along with RNA polymerase II, are translocated to these RCs to facilitate active gene expression. The movement of small RCs is also aided by actin and myosin [72, 74].

Within the nuclear RCs, the assembly of progeny virions takes place. This encompasses capsid formation and the packaging of viral DNA. An internal scaffold is formed within these compartments to aid in capsid assembly and genome incorporation. After assembly, the nucleocapsids exit the nucleus through a budding mechanism involving the nuclear membrane. Subsequently, mature virions are transported to the host cell’s plasma membrane, from which they are released through membrane fusion [75].

HSV has evolved strategies to manipulate host cell defenses to its advantage. One such defense is the formation of Promyelocytic Leukemia Nuclear Bodies (PML-NBs), which are involved in the cellular antiviral response. HSV disrupts PML-NBs, creating a more favorable environment for viral replication. Furthermore, the virus modulates the cellular DNA damage response (DDR). While some DDR components are beneficial for viral replication, specific viral proteins, such as ICP0, target and degrade others involved in sensing DNA damage. This manipulation allows HSV to harness the DDR for its replication needs [72].

Various antiviral strategies are employed against HSV infections. Receptor-targeting therapeutics prevent viral binding to host cells, including drugs like anti-heparan sulfate peptides, apolipoprotein E, and AC-8. Nucleic acid-based molecules like aptamers and dermaseptins are also used. Viral glycoprotein-targeting therapeutics, such as nanoparticles, K-5, SP-510-50 compounds, and dendrimers, inhibit viral fusion and replication. Targeting downstream signaling cascades focuses on inhibiting kinases like PI3K. These approaches aim to enhance the management of HSV infections, with ongoing studies to assess their effectiveness [76].

4. Conclusion

The intricate dance between viruses and host cells has long fascinated researchers, centering on the phenomenon of virus replication cycles, which are fundamental to viral infections. These cycles reveal how viruses infiltrate, replicate, and disseminate, enriching our understanding of viral biology. RNA and DNA viruses, with distinct strategies, pose unique challenges in understanding and combating infections. By studying virus replication cycles and delving into their mechanisms, significance, and their impact on disease, we gain deeper insights into how viruses shape infection outcomes. Understanding virus replication cycles is critical for advancing our knowledge of infectious diseases and developing effective strategies to combat them.

As we’ve explored examples or different RNA and DNA viruses, we’ve encountered a fascinating array of mechanisms governing their replication which is vital for developing effective strategies against them.

RNA viruses, known for their rapid changes and mutations, pose challenges in drug development. They use RNA-dependent RNA polymerase (RdRp) for replication, making RdRp a promising target. However, because RNA viruses change quickly, we need adaptable approaches. On the other hand, DNA viruses have more stable genomes and use DNA polymerases in their replication, giving us different targets to consider.

RNA viruses, like hepatitis C, influenza A, coronaviruses, and so on, possess single-stranded RNA genomes. Their replication cycles involve unique strategies, including RNA-dependent RNA polymerases, viral RNA integration into host DNA (as seen in HIV), and frequent mutations contributing to their adaptability and the challenge they pose to antiviral development.

On the other hand, DNA viruses, exemplified by Adenovirus, Papillomaviruses, Poxviridae, Simplex Viruses, and so forth, employ diverse tactics. They may initiate replication at specific origin sites within their genomes (like EBV), manipulate host defenses (as observed in HSV), or modulate the cellular DNA damage response (as in Adenovirus). These viruses intricately interact with the host machinery to ensure their survival and propagation.

Research into these replication cycles is paramount for several reasons. First, gaining knowledge about these procedures offers valuable insights into viral pathogenesis and contributes to the advancement of efficient antiviral treatments and vaccine development. Second, as evidenced by the pandemic of COVID-19, induced by Coronaviruses, a deeper comprehension of virus replication is vital for pandemic preparedness and response. Lastly, as we continue to discover new viruses and variants, research into their replication cycles remains at the forefront of virology.