Positive-strand (+)-RNA viruses rely on a unique replication mechanism that directly harnesses their RNA genomes as templates for protein synthesis¹. These viruses, such as SARS-CoV-2, hepatitis C virus (HCV), chikungunya virus (CHIKV), and flaviviruses, possess single-stranded RNA genomes that function similarly to messenger RNA (mRNA) upon entering host cells. This allows them to be immediately translated by the host’s ribosomes into proteins essential for virus replication.

Once inside the cell, one of the first tasks of these viruses is to reorganize internal membranes to build structures known as replication organelles (ROs). These specialized compartments not only shelter the viral replication machinery from the host’s immune defenses but also concentrate necessary lipids and enzymes, creating an optimized environment for viral RNA synthesis. For example, coronaviruses reshape the endoplasmic reticulum (ER) into double-membrane vesicles where replication occurs.

A key enzyme in this process is RNA-dependent RNA polymerase (RdRp), which copies the viral genome by synthesizing a complementary negative-strand RNA. This negative strand then serves as a template to generate more (+)-RNAs, fueling the production of viral proteins and new virions. In addition, different virus families manage their protein expression in diverse ways: some translate a single polyprotein that is later cleaved into functional units (such as picornaviruses), while others (such as coronaviruses and togaviruses) use subgenomic RNAs to control the expression of structural proteins.
Although all (+)-RNA viruses depend on similar molecular strategies, they differ in how they remodel host membranes and regulate replication timing. Despite their small genomes, these viruses show remarkable efficiency and flexibility in hijacking cellular pathways, making them both scientifically intriguing and clinically important.

Understanding these replication processes continues to inform the development of targeted antiviral drugs and vaccine platforms, offering hope for mitigating the diseases they cause.

Key enzymes in positive-strand RNA replication

Key enzymes required for (+)-RNA replication include RNA polymerases, helicases, and primases. Each is dedicated to facilitating transcription as well as replication of the RNA genomes in both the virus and host cells. Some essential enzymes involved in RNA replication are:

• RdRp: RdRp polymerase is the central enzyme involved in RNA replication. It catalyzes the synthesis of RNA from an RNA template². In the case of (+)- RNA viruses, RNA polymerase synthesizes viral RNA genomes, which can act as messenger RNA (mRNA), which host cell ribosomes translate to produce viral proteins. This enzyme is used by RNA viruses such as hepatitis C, Zika virus, and coronaviruses to replicate RNA from an RNA template.
• Helicase: Helicase enzymes are necessary for unwinding the double-stranded regions of RNA during replication. These enzymes work by breaking the hydrogen bonds between complementary RNA strands, allowing the RNA polymerase to access the template strand and synthesize a new RNA molecule³.
  Helicases are involved in both DNA and RNA replication but are particularly important in the replication of RNA viruses, where the RNA genome can exist in various secondary structures. In coronaviruses such as SARS-CoV-1 and SARS-CoV-2, the NSP13 gene encodes a helicase enzyme. This helicase is crucial for viral replication owing to its role in unwinding RNA. Similarly, HCV utilizes its NS3 gene to produce a helicase. This enzyme operates in a 3’–5’ antisense direction along the RNA, facilitating processes essential for viral replication.

Role of RNA polymerase in replicating positive-strand RNA

RNA polymerase plays a vital role in synthesizing RNA molecules during RNA replication. It initiates RNA synthesis either:

• De novo synthesis (without a primer), wherein RdRp initiates RNA synthesis without a pre-existing primer; ie, it starts directly from a single nucleotide, which provides the 3’-hydroxyl group for reaction initiation, such as in Flaviviridae.
• Or in some cases, a viral protein genome-linked protein (VPg) is used to initiate RNA synthesis by the RdRp. An example is the Picornaviridae family (eg, poliovirus).

Overview of the replication cycle of positive-strand RNA viruses

Viral entry: The replication cycle begins when the virus binds to specific receptors on the host cell surface. Entry mechanisms vary among viruses. Many, such as the dengue virus and poliovirus, use receptor-mediated endocytosis to enter the cell. Enveloped viruses, such as coronaviruses, fuse with the plasma membrane or endosomal membrane, and non-enveloped viruses, such as norovirus, create pores in the endosomal membrane to inject their RNA genome directly into the cytoplasm. Once inside, the viral capsid disassembles, releasing the RNA genome into the cytoplasm.

Immediate translation of viral genome: Because the (+)-RNA genome functions as an mRNA, host ribosomes immediately begin translating it into viral proteins. Most (+)-RNA viruses produce a single large polyprotein, which is later cleaved into functional viral proteins by viral or host proteases. For example, the poliovirus synthesizes a polyprotein that is processed into structural and nonstructural proteins (NSPs), including RdRp. Some flaviviruses, such as Zika and dengue viruses, also produce subgenomic RNAs that regulate gene expression. Among the translated proteins, the RdRp plays an important role in RNA replication.

Cleavage of polyproteins to functional proteins: Because polyproteins are not functional until they are cleaved, viral and host proteases play an important role in converting them into active proteins. Many (+)-RNA viruses produce proteases, which cleave the polyprotein at specified places. For example, the 3C protease of picornaviruses cleaves many sites in the polyprotein to produce functional viral proteins, while flaviviruses contain an NS2B-NS3 protease that processes the polyprotein at particular cleavage sites.

Replication initiation: After translation, the viral RdRP initiates genome replication by synthesizing a complementary negative-strand RNA using the (+)-RNA as a template. This negative-strand RNA then serves as a template for generating multiple new (+)-RNA genomes. Replication often occurs within the specialized membrane-bound ROs, which are formed by modifying a diverse array of host organelle membranes.

Viral ROs are subcellular compartments produced from the host cell’s organelle membranes that facilitate viral replication to occur. ROs concentrate viral genomes, replicase proteins, and other host components, which improves replication efficiency. They protect viral replication intermediates from host immune responses, such as RNA silencing and interferon induction. These also generate a protected milieu for viral genome replication, which leads to the formation of new virions.

Many (+)-RNA viruses modify the ER to create replication sites. Some viruses, such as coronaviruses and picornaviruses, cause the formation of double-membrane vesicles (DMVs; 200–300 nm in diameter) from the internal membranes of virus-infected cells. Viruses can also employ other organelles, including the Golgi apparatus and mitochondria, depending on the viral type.
Coronaviruses and flaviviruses replicate within vesicles originating from the ER⁴, while nodaviruses utilize the outer mitochondrial membrane as their replication site. In contrast, picornaviruses modify Golgi membranes to support replication. These specialized structures shield viral RNA from host immune defenses and enhance replication efficiency.

Figure: Common membranous structures occurring in host cells with positive-strand RNA viruses.

This image is from an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted, which does not comply with these terms⁵. Reference: Nguyen-Dinh V, Herker E. Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses. Cells. 2021; 10(9):2407. https://doi.org/10.3390/cells10092407

(+)-RNA viruses encode NSPs that play a critical role in replication complex formation and function. For example:

• NSPs help localize RdRp and viral RNA templates within ROs, and picornavirus 3D polymerase (3Dpol) and flavivirus NS5 RdRp are crucial for RNA synthesis.
• Poliovirus proteins 2BC and 3A disrupt Golgi membranes, while flavivirus proteins NS4A and NS4B modify ER membranes⁶.
• To provide the building blocks required for membrane remodeling, several viral NSPs interact with host proteins involved in lipid metabolism⁷.

NSPs from SARS-CoV-2 suppress innate immune responses while facilitating replication complex formation.

Packaging of the viruses: Newly synthesized (+)-RNA genomes are then packaged into viral capsids along with structural proteins. The site of viral assembly varies among different viruses. Non-enveloped viruses, such as poliovirus and norovirus, assemble their virions directly in the cytoplasm. Enveloped viruses, such as dengue and hepatitis C viruses, assemble within the ER or Golgi complex before being transported to the cell membrane for release by exocytosis. Some viruses also incorporate host-derived lipid membranes during this process, aiding in immune evasion.

Viral release: The final step in the replication cycle is the release of mature virions from the host cell. Non-enveloped viruses, such as poliovirus, typically cause cell lysis, leading to the release of large numbers of virions at once. Enveloped viruses, such as SARS-CoV-2, flaviviruses (eg, dengue; Zika), and togaviruses (eg, Chikungunya; Sindbis virus), exit the cell through exocytosis, allowing continuous virus release without immediately killing the host cell^{6, 7}. Once released, these new virions infect neighboring cells and repeat the replication cycle.

Table: Examples of (+)-RNA viruses and their replication characteristics:

Nodavirus as a model positive-strand RNA virus

Nodaviruses belong to the Nodaviridae family and are small (+)-RNA viruses that infect both vertebrates and invertebrates6. Their simple genome and single RNA replication protein, protein A, make them an excellent model for studying viral RNA replication. Protein A localizes to the outer mitochondrial membrane, where it induces the formation of ROs. ROs then facilitate efficient viral RNA synthesis and serve as platforms for genome amplification.
Nodaviruses also possess a unique RNA capping mechanism, similar to alphaviruses, but their polymerase structure resembles that of flaviviruses, positioning them as an evolutionary link between these viral families. The best-studied nodavirus, Flock House virus (FHV), replicates in a remarkably broad range of host cells, making it a versatile tool for research^{6, 7, 8}.

Regulatory mechanisms in positive-strand RNA replication

Proteins found in host cells can either facilitate or inhibit viral RNA replication, altering viral propagation efficiency^{8, 9}. Furthermore, viral regulatory proteins can influence the host cell machinery, ensuring optimal replication circumstances.

• Host cellular factors: Some of the host cell proteins involved in RNA replication include helicases, chaperones, and transcription factors. Some viruses hijack these proteins to optimize the replication of viral RNA, which is often important for successful infection and replication.
• Viruses often exploit host RNA helicases to unwind viral RNA secondary structures, enhancing replication efficiency^9–11. For example, HCV recruits the host’s DEAD-box helicase, DDX3X, to facilitate the replication of its RNA genome. Similarly, coronaviruses, including SARS-CoV, have been shown to interact with host helicases such as DDX1 and DDX5 to promote viral RNA synthesis.

Viruses use host chaperones to ensure the correct folding of their replication machinery¹². For example, Tombusvirus spp. recruit the host’s Hsp70 chaperone to their replication sites, aiding in the assembly of functional viral replicase complexes. This interaction is vital for efficient viral RNA replication.

Transcription factors and regulatory proteins

Host transcription factors in viral replication

In some instances, the replication of RNA viruses is controlled by cellular transcription factors that regulate the expression of host genes. (+)-RNA viruses do not use host transcription machinery for genome synthesis and instead regulate transcription factors to control immune evasion, stress responses, and metabolism. Viruses such as HCV and SARS-CoV-2 stimulate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which promotes pro-virus inflammation. The dengue virus increases the rate of replication and decreases the production of IFN-β by inhibiting IRF3, which is essential for interferon responses. HCV promotes viral replication in hepatocytes by stabilizing HIF-1α, which improves lipid metabolism and glycolysis. Through these associations, viruses can enhance cellular conditions for replication and evade immune responses.

Viral proteins regulating host protein functions

Viruses carry the potential to manipulate host transcription factors by enhancing their own replication¹¹. One example is the human immunodeficiency virus type 1 (HIV-1) Tat protein, which amplifies viral transcription by interacting with host cells¹³. Some immune factors of the host, including interferons, can be used to inhibit viral RNA replication and trigger antiviral responses. (+)-RNA viruses use NSPs to influence host pathways during replication. For instance, coronavirus NSP1 destroys host mRNA to ensure viral RNA translation; HCV NS5A inhibits apoptosis via interacting with the PI3K/AKT signaling pathway; Flavivirus NS5 (eg, Dengue, Zika, and West Nile Virus) suppresses STAT2, preventing interferon activation and allowing immune evasion, thereby enhancing viral persistence.

Applications

The study of RNA replication has profound implications in fields such as viral disease and biotechnology. This understanding is vital for generating antiviral medicines, designing vaccines, and discovering new uses in molecular biology.
Significance in viral replication and infectious diseases: RNA replication² is central to the multiplication of RNA viruses that infect human beings, animals, and plants. The study of (+)-RNA replication may reveal many possible antiviral drug targets, such as inhibitors of RNA polymerases or viral replication complexes. Improving our understanding of how RNA viruses replicate can also bolster the development of vaccines that target these processes, providing effective strategies for preventing and controlling viral infections.

Biotechnology and medicine

RNA vaccines: mRNA vaccines are prepared by inserting a piece of messenger RNA that corresponds to the viral proteins. Self-amplifying mRNAs derived from (+)-RNA viruses show promise as vaccination platforms. These vaccines simulate viral infection by amplifying their genome and antigen-encoding mRNA in host cells, resulting in prolonged protein production, innate immune activation, and robust, long-lasting humoral and cellular immune responses. For example, the COVID-19 mRNA vaccines are among the most recent RNA-based therapeutic advances^14–16. These vaccines work on the principle of synthetic mRNA that encodes the spike protein of the SARS-CoV-2 virus. Once administered to the body, the COVID-19 mRNA induces the cell machinery to produce the viral spike protein, thereby triggering an immune response without leading to the disease.

These vaccines are taken up by immune or non-immune cells, which translate the mRNA into antigens that activate T and B cells. However, mRNA molecules are unstable and susceptible to degradation by nucleases¹³. Encapsulation within liquid nanoparticles (LNPs) shields mRNA from enzymatic degradation, enhancing its stability during delivery¹⁵.
mRNA vaccines have also shown their potential for the rabies virus and COVID-19¹⁷. For example, a non-replicating mRNA vaccine encoding the rabies virus glycoprotein (rabies-G) has demonstrated the ability to induce neutralizing antibodies lasting up to one year in animal models. Additionally, some vaccines have undergone phase 1 clinical trials and are reported to be well-tolerated and capable of inducing robust immune responses in healthy adults.

• Synthetic biology and bioengineering: (+)-RNA replication is being harnessed in synthetic biology to develop programmable biological systems. Synthetic biology helps to increase the stability of mRNA vaccines by addressing problems such as a short lifespan in physiological circumstances. Due to codon optimization, nucleic acid modifications, and polyadenylation, modified RNA exhibits improved durability and production of mRNA vaccines after injection, and increases their effectiveness.
• Gene therapy: RNA replication is involved in gene therapy, in which the RNA molecules are delivered to the cells for the correction of genetic diseases18. (+)-RNA viruses have contributed to the development of RNA-based therapies, particularly in vaccine technology and transient gene expression systems. One promising approach involves the use of self-replicating RNA (replicon) vectors derived from (+)-RNA viruses, such as alphaviruses (eg, Sindbis virus). Among self-replicating (+)-RNA viruses, alphaviruses, and flaviviruses have been used for vaccine development and gene therapy. These viruses are capable of high-level RNA amplification directly in the cytoplasm of host cells. For example, alphaviruses can accumulate approximately 1x10⁶ RNA copies per cell. In addition to amplifying large quantities of RNA copies, the expression levels of the gene of interest are very high, as the expression is driven by strong viral promoters.

RNA-based approach can be used on antisense oligonucleotides (ASOs) to modulate gene expression of viruses19. ASOs are short, synthetic strands of nucleotides designed to bind specific mRNA sequences, thereby influencing their splicing or stability. For instance, peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) ASOs have been shown to effectively inhibit flavivirus infections (West Nile virus; dengue virus) by targeting conserved sequences within the 5’- and 3’-untranslated regions (UTRs) of the viral genome.

RNA replication research has led to innovative cancer treatments, including RNA-based cancer vaccines and RNA interference (RNAi)-based therapies. RNA-based cancer vaccines work by introducing synthetic mRNA encoding tumor-associated antigens. These vaccines train the immune system to recognize and destroy cancer cells. For example, synthetic mRNA vaccines targeting melanoma and lung cancer have shown promising results in clinical trials²⁰. In animal models, (+)-strand RNA viruses are being studied and used for cancer treatment, specifically as oncolytic viruses, as well as the development of vaccinations, with some showing promising results in preclinical and clinical trials. Certain (+)-RNA viruses are being modified to solely infect and destroy cancer cells, leaving healthy organs unaffected. For example, synthetic RNA viruses derived from picornaviruses are being created to systematically deliver viral RNA to tumor cells²¹.

Newcastle disease virus, a well-known RNA virus, has oncolytic capabilities, exhibiting anticancer effects²¹.
Poliovirus has been proven in animal models to efficiently replicate, leading to tumor regression²².
RNAi is another approach where small interfering RNAs (siRNAs) or microRNAs (miRNAs) silence the expression of oncogenes23. These RNA molecules bind to target mRNA sequences, leading to mRNA degradation or inhibition of translation and, consequently, inhibition of the production of proteins that promote tumor growth. Positive-strand RNA viruses-based RNAi therapies are currently being tested for liver cancer, leukemia, and glioblastoma. For example, lentiviruses offer the potential to transduce stem cells, making them particularly attractive tools for the delivery of RNAi to these non-proliferating or slowly proliferating cell types²⁴.

Understanding (+)-RNA replication mechanisms has facilitated the development of antiviral drugs targeting these viruses. One example is remdesivir, a nucleoside analog that inhibits the RNA-dependent RNA polymerase of SARS-CoV-2, preventing viral replication²⁵.

Other antiviral strategies include:

• RNA aptamers: short RNA sequences that bind to viral proteins and block replication. The potential of aptamers in the diagnosis and treatment of viral illnesses, including AIDS, hepatitis B and C, SARS, and Ebola, is being studied. According to recent studies, aptamers that target the receptor binding region of the SARS-CoV-2 spike protein show potential to be used as COVID-19 diagnostic and treatment tools²⁶.
• CRISPR-Cas13, an RNA-targeting system that degrades viral RNA, offers a potential antiviral strategy against diseases such as influenza and COVID-19.
  These approaches are being explored for treating RNA virus infections such as hepatitis C, Zika, and Ebola²⁷.

In agriculture and plant biotechnology, research on (+)-RNA replication is also transforming agriculture by developing RNA-based pesticides and antiviral treatments for crops. RNAi²³ technology is being used to target plant viruses, pests, and fungal pathogens without harming beneficial organisms. For instance, several RNA virus vectors, such as tobacco rattle virus (TRV), potato virus Y (PVY), TMV, and PLRV, have been successfully used for virus-induced gene silencing²⁸. The use of infectious RNA virus clones has facilitated targeted gene amplification and provided a convenient vector platform that can circumvent RNAi for site-directed mutations while increasing or decreasing gene expression to characterize PTGS and produce valuable heterologous commercial products.

Challenges and future directions in positive-strand RNA replication research

Research into positive-strand RNA [(+)-RNA] virus replication continues to uncover complex challenges, particularly in understanding the role of host cell membranes. A key focus is how lipid composition and membrane dynamics influence the formation and stability of replication organelles (ROs). These specialized membrane structures are essential for viral replication, yet the precise mechanisms behind their biogenesis remain unclear. Investigating how host and viral proteins interact to remodel membranes and initiate RO formation is crucial. Additionally, the roles of lipid transfer proteins and membrane contact sites in facilitating RO development are emerging areas of interest. Another critical challenge is determining how ROs evade or modulate host immune responses, allowing viruses to persist and replicate efficiently.

Future research on (+)-RNA virus replication is expected to delve deeper into host-virus interactions, particularly how these viruses evade immune detection and contribute to disease. A promising direction involves studying the mechanisms of RO formation, including their lipid composition and the regulation of lipid fluxes. Advancements in reverse genetics could revolutionize our understanding of viral gene function and support the development of novel antiviral therapies. Targeting ROs directly presents a compelling therapeutic strategy, as disrupting these structures could inhibit viral replication. Additionally, exploring methods to interfere with viral RNA replication and translation remains vital for therapeutic innovation.

FAQs

What are the main stages of positive-strand RNA replication?

Positive-strand RNA virus replication occurs through the following key steps. First, the virus binds to specific host cell receptors before entering through endocytosis or membrane fusion. Once within, the viral DNA is rapidly translated into proteins, including those necessary for replication. These viral proteins then alter host membranes, resulting in specialized ROs. Within these compartments, the viral RNA-dependent RNA polymerase (RdRp) produces new genomic RNA. Finally, newly formed virions are packaged and removed from the host cell by lysis or exocytosis.

How does positive-strand RNA replication differ from DNA replication?

Positive-stranded RNA replication differs from DNA replication in many ways. Positive-strand RNA viruses replicate entirely in the cytoplasm, whereas DNA replication happens in the nucleus and requires a DNA template. These viruses do not need a DNA intermediary or host polymerases; instead, they use membrane-bound replication complexes to generate new RNA genomes straight from the viral RNA template.

What is the significance of the promoter region in positive-strand RNA replication?

The promoter region controls viral RNA synthesis by directing RdRp to certain sequences (+)-strand RNA replication. This sequence is found in the 5’' and 3’' untranslated regions, or internal sites, where it draws host and viral factors to ensure successful replication, genome amplification, and the proper balance of subgenomic RNA transcription and genome replication.

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