# Lecture 15: Virology II -- Replication Strategies
## Microbiology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Describe the general steps of the viral replication cycle
2. Compare replication strategies of the major Baltimore classes
3. Explain the replication cycle of a representative DNA virus (herpesvirus), RNA virus (influenza), and retrovirus (HIV)
4. Define and distinguish between the lytic and lysogenic cycles
5. Explain how viruses cause cell damage and the outcomes of viral infection
6. Describe viral oncogenesis
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## Lecture Content
### I. General Steps of the Viral Replication Cycle
The viral replication cycle proceeds through six general steps, each of which is essential for the production of new virions.
**Attachment (adsorption)** is the first step, in which viral attachment proteins (VAPs) bind specifically to receptors on the host cell surface. This interaction determines both tropism and host range. Examples of well-characterized attachment events include HIV gp120 binding to CD4 plus a coreceptor (CCR5 or CXCR4), influenza hemagglutinin binding to sialic acid residues, and the SARS-CoV-2 spike protein binding to ACE2. In non-enveloped viruses, capsid proteins directly mediate attachment.
**Penetration (entry)** follows attachment. Enveloped viruses enter by membrane fusion, which can occur either at the plasma membrane (triggered by receptor binding, as in HIV) or within an endosome after endocytosis (triggered by low pH, as in influenza). Non-enveloped viruses typically enter by receptor-mediated endocytosis followed by lysis of the endosome, or by direct injection of the genome through a pore.
**Uncoating** is the release of viral nucleic acid from the capsid into the cytoplasm or nucleus. This may occur simultaneously with penetration or after endosomal processing.
**Biosynthesis (genome replication and gene expression)** is the central phase, and its specific strategy depends on the type of nucleic acid and thus the Baltimore class of the virus. Early genes typically encode enzymes needed for genome replication and may shut off host macromolecular synthesis. Late genes encode structural proteins such as capsid subunits and envelope glycoproteins.
**Assembly (morphogenesis)** involves the self-assembly of capsid proteins around the newly synthesized genomes. For most RNA viruses, assembly occurs in the cytoplasm, while most DNA viruses assemble in the nucleus (with the notable exception of poxviruses, which assemble in the cytoplasm). In enveloped viruses, viral glycoproteins are inserted into host membranes at the ER, Golgi, or plasma membrane.
**Release** is the final step. Non-enveloped viruses are typically released by lysis of the host cell. Enveloped viruses exit by budding from host cell membranes, acquiring their envelope in the process; the cell may survive initially during this slow release but eventually dies. In the case of influenza, neuraminidase cleaves sialic acid residues to release progeny virions from the cell surface, a step targeted by the antiviral drug oseltamivir (Tamiflu). The number of progeny virions produced per cell, known as the burst size, varies from approximately 10 to more than 10,000 depending on the virus.
A step-by-step diagram of the generalized viral replication cycle. Six numbered steps arranged around a host cell: (1) Attachment -- virus binding to a surface receptor; (2) Penetration -- enveloped virus fusing with the plasma membrane, and non-enveloped virus entering via endocytosis (two pathways shown); (3) Uncoating -- release of nucleic acid from the capsid inside the cell; (4) Biosynthesis -- genome replication and mRNA transcription in the nucleus or cytoplasm (with early and late gene products labeled); (5) Assembly -- capsid proteins assembling around new genomes; (6) Release -- non-enveloped virus by cell lysis, enveloped virus by budding (with glycoprotein spikes being acquired from the host membrane). Arrows show the direction of the cycle.
### II. Replication Strategies by Baltimore Class
#### A. Class I: dsDNA Viruses
Class I viruses with double-stranded DNA genomes generally replicate in the **nucleus**, with the important exception of poxviruses, which replicate in the cytoplasm and carry their own transcription machinery. Most dsDNA viruses use the host RNA polymerase for mRNA transcription (as do herpesviruses, adenoviruses, and papillomaviruses), though some encode their own DNA polymerase -- the herpesvirus DNA polymerase, for example, is the target of acyclovir.
The **herpesvirus replication cycle** illustrates this class well. Upon infection, the viral envelope fuses with the plasma membrane, and the nucleocapsid is transported to a nuclear pore, where the linear dsDNA enters the nucleus and circularizes. Transcription proceeds in a regulated temporal cascade: immediate-early (alpha) genes are transcribed first by host RNA Pol II and encode regulatory proteins. Early (beta) genes follow, encoding enzymes needed for DNA replication, including viral DNA polymerase and thymidine kinase. DNA replication proceeds by a rolling circle mechanism, producing concatemeric DNA. Late (gamma) genes encode structural proteins, including capsid components and envelope glycoproteins. Capsids are assembled in the nucleus and loaded with DNA. The nucleocapsid then buds through the inner nuclear membrane to acquire a primary envelope, de-envelopes, and re-envelopes at the Golgi or trans-Golgi network before being released by exocytosis.
A defining feature of herpesviruses is **latency**: the viral genome persists as a circular episome in the nucleus with limited gene expression and can reactivate under appropriate stimuli. HSV-1 and HSV-2 establish latency in sensory neurons of the trigeminal and sacral ganglia, respectively. VZV lies latent in dorsal root ganglia and reactivates to cause shingles. EBV persists in B lymphocytes.
#### B. Class IV: (+)ssRNA Viruses
Positive-sense single-stranded RNA viruses have genomes that are directly translatable as mRNA upon entry into the host cell, so there is no need to carry a polymerase in the virion. Replication occurs entirely in the **cytoplasm**. The viral RNA-dependent RNA polymerase (RdRp) is translated from the genome and then replicates the RNA through a negative-sense intermediate: (+)RNA is copied to (-)RNA (the replicative intermediate), which in turn serves as a template for new (+)RNA genomes and mRNA molecules.
**Poliovirus** (a picornavirus) exemplifies this class. Its genome is a single (+)ssRNA molecule with a 5' VPg protein (rather than a cap) and a 3' poly-A tail. The entire genome is translated as one large polyprotein, which is then cleaved by viral proteases into individual functional proteins. The viral RdRp (3Dpol) replicates the genome. Assembly occurs in the cytoplasm, and progeny virions are released by cell lysis.
**Coronaviruses**, including SARS-CoV-2, possess the largest known RNA virus genomes at approximately 30 kb. The 5' two-thirds of the genome is translated into polyproteins pp1a and pp1ab, which are processed to form a replicase complex containing the RdRp, helicase, and a proofreading exonuclease (an unusual feature among RNA viruses). Subgenomic mRNAs are produced for the structural proteins (S, E, M, N). Assembly occurs at the ER-Golgi intermediate compartment (ERGIC), and virions are released via exocytosis.
#### C. Class V: (-)ssRNA Viruses
Negative-sense single-stranded RNA viruses carry genomes that **cannot** serve as mRNA because they are the antisense complement of the message. Consequently, they must carry an **RdRp in the virion** to transcribe (-)RNA into (+)mRNA immediately upon entry.
**Influenza virus** (Orthomyxoviridae) is a well-studied example. Its genome consists of 8 segmented (-)ssRNA molecules, each encoding one to two proteins. Unusually for an RNA virus, replication occurs in the **nucleus**. The viral RdRp, composed of PA, PB1, and PB2 subunits, transcribes mRNA using a process called "cap snatching," in which it steals 5' caps from host pre-mRNAs to prime viral mRNA synthesis. The same RdRp also replicates full-length (-)RNA via a (+)RNA intermediate called cRNA. The segmented genome enables **reassortment** (antigenic shift) when two different influenza strains co-infect the same cell and exchange genome segments.
**Non-segmented (-)ssRNA viruses**, including members of the Paramyxoviridae, Rhabdoviridae, and Filoviridae, replicate in the cytoplasm. Their RdRp transcribes mRNA from the 3' end of the genome in a sequential, gradient fashion, so that promoter-proximal genes are transcribed at higher levels.
#### D. Class VI: Retroviruses (HIV)
Retroviruses carry **two copies** of a (+)ssRNA genome in each virion, making them diploid. They also package **reverse transcriptase (RT)**, integrase, and protease within the virion.
The replication cycle of **HIV** proceeds as follows. The surface glycoprotein gp120 binds CD4 on T helper cells, and gp41 then mediates membrane fusion, a process that also requires engagement of a coreceptor (CCR5 or CXCR4). After the nucleocapsid enters the cytoplasm, reverse transcriptase converts the ssRNA genome to dsDNA (proviral DNA). RT possesses RNase H activity to degrade the RNA template during this process. Importantly, RT is error-prone and lacks proofreading, resulting in a high mutation rate that drives antigenic variation and drug resistance. The proviral dsDNA enters the nucleus, where integrase inserts it into the host chromosome, creating a provirus. The proviral DNA is then transcribed by host RNA Pol II to generate viral mRNA and genomic RNA. These mRNAs are translated into polyproteins (Gag, Gag-Pol, Env). Assembly occurs at the plasma membrane, where immature virions bud and acquire an envelope studded with gp120/gp41. The viral protease cleaves the polyproteins during or after budding in a maturation step that yields the fully infectious virion.
The multiple steps of the HIV replication cycle provide numerous drug targets: RT is targeted by NRTIs (AZT, tenofovir) and NNRTIs (efavirenz), the protease by protease inhibitors (ritonavir, darunavir), integrase by integrase inhibitors (dolutegravir, raltegravir), and the entry/fusion process by entry inhibitors (maraviroc) and fusion inhibitors (enfuvirtide).
A detailed diagram of the HIV replication cycle inside a CD4+ T cell. Step 1: gp120 binds CD4 and CCR5/CXCR4 coreceptor; gp41 mediates fusion. Step 2: Uncoating releases RNA genome and RT into the cytoplasm. Step 3: RT converts ssRNA to dsDNA (showing the RNA/DNA hybrid intermediate and RNase H activity). Step 4: dsDNA enters nucleus; integrase inserts provirus into host chromosome. Step 5: Host RNA Pol II transcribes proviral DNA into viral mRNA and genomic RNA. Step 6: mRNA exported to cytoplasm; translated into Gag, Gag-Pol, and Env polyproteins. Env processed in ER/Golgi. Step 7: Assembly at plasma membrane; budding. Step 8: Protease cleaves Gag and Gag-Pol polyproteins (maturation). Drug targets annotated at each relevant step (NRTI, NNRTI, integrase inhibitor, protease inhibitor, entry inhibitor).
#### E. Class VII: dsDNA-RT Viruses (Hepadnaviruses -- HBV)
Hepatitis B virus has a partially double-stranded circular DNA genome known as relaxed circular DNA (rcDNA). In the nucleus, rcDNA is repaired to form **covalently closed circular DNA (cccDNA)**, which serves as the template for all viral transcription. A pregenomic RNA (pgRNA) is transcribed from cccDNA and then reverse transcribed by the viral RT back into DNA within newly assembled capsids. The persistence of cccDNA in hepatocytes makes HBV extremely difficult to eradicate and underlies chronic infection. Antiviral agents targeting the viral RT, such as tenofovir and entecavir, are used for treatment.
### III. Outcomes of Viral Infection at the Cellular Level
Viral infection can produce several distinct outcomes at the cellular level. **Lytic infection** involves rapid viral replication, production of progeny virions, and killing of the host cell, with cytopathic effects including cell rounding, lysis, and plaque formation. **Persistent infections** take several forms: **chronic (productive) infections** involve continuous low-level virus production while the cell survives (as seen with HBV and HCV); **latent infections** involve the presence of the viral genome with minimal or no gene expression and no virus production, but with the potential for reactivation (herpesviruses, HIV provirus); and **slow infections** have long incubation periods of months to years before progressive disease develops (HIV progressing to AIDS, prion diseases). **Transforming (oncogenic) infections** alter cellular growth control and can lead to cancer. **Abortive infections** occur when a virus enters a cell but cannot complete its replication cycle because the cell type is restrictive.
### IV. Cytopathic Effects (CPE) and Cell Damage
Viruses damage host cells through multiple mechanisms. They may inhibit host DNA, RNA, or protein synthesis by shutting off host macromolecular machinery. Accumulation of viral proteins can disrupt cellular membranes, leading to lysis. Many viruses induce **apoptosis** (programmed cell death). Some viruses cause the formation of **syncytia** -- multinucleated giant cells produced by fusion of neighboring cells -- as seen with HIV, measles, RSV, and herpesviruses. **Inclusion bodies**, which are aggregates of viral components visible by light microscopy, serve as important diagnostic markers: intranuclear inclusions include Cowdry type A bodies (herpesvirus) and owl's eye inclusions (CMV), while intracytoplasmic inclusions include Negri bodies (rabies) and Guarnieri bodies (smallpox). Beyond direct viral damage, the immune response itself can cause significant tissue injury through the actions of cytotoxic T lymphocytes, antibody-dependent mechanisms, and inflammatory responses. Hemorrhagic fever viruses produce vascular damage and hemorrhage.
### V. Viral Oncogenesis
**Oncogenic viruses** contribute to an estimated 15--20% of human cancers through several mechanisms. Some carry viral oncogenes that directly stimulate cell proliferation, such as v-src in Rous sarcoma virus. Others encode proteins that inactivate tumor suppressors: HPV E6 degrades p53, HPV E7 inactivates Rb, and SV40 large T antigen binds both p53 and Rb. Insertional mutagenesis occurs when a provirus integrates near a proto-oncogene and activates its expression. Chronic inflammation and sustained cell turnover, as caused by HBV and HCV, can lead to hepatocellular carcinoma over decades. Immunosuppression by viruses such as HIV increases the risk of cancers driven by other oncogenic viruses, including Kaposi sarcoma (HHV-8) and lymphomas (EBV).
The key oncogenic viruses in humans include **HPV** (cervical, oropharyngeal, and anal cancer), **EBV** (Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin lymphoma), **HHV-8** (Kaposi sarcoma), **HBV and HCV** (hepatocellular carcinoma), **HTLV-1** (adult T-cell leukemia/lymphoma), and **Merkel cell polyomavirus** (Merkel cell carcinoma).
A diagram summarizing viral oncogenesis mechanisms. Panel A: HPV oncoproteins -- E6 binding and targeting p53 for proteasomal degradation via ubiquitin ligase E6AP; E7 binding and inactivating Rb, releasing E2F transcription factor to drive cell cycle progression. Both pathways converge on uncontrolled cell proliferation. Panel B: Retroviral insertional mutagenesis -- provirus integrating upstream of a proto-oncogene (e.g., c-myc), with the viral LTR promoter driving constitutive overexpression. Panel C: Chronic inflammation model -- HBV/HCV causing chronic hepatitis, repeated cycles of liver cell death and regeneration, accumulation of mutations over decades, leading to hepatocellular carcinoma. Each panel labeled with the virus, mechanism, and associated cancer.