[Chinese Academy of Sciences] Molecular Biology - Zhu Yuxian Fourth Edition - Notes - Molecular Biology of Viruses

Molecular Biology of Viruses

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12. Molecular biology of viruses (Chapter 9 of the textbook)

12.1 Basic Concepts of Viruses

● Concepts related to infectious diseases

• Pathogen : An organism that causes disease in the human body.
• Bacteria and viruses are the best known pathogens
• Fungi, protozoa, and parasites also cause disease: tinea corporis, malaria, angiostrongyliasis cantoniasis, etc.
• Infectious disease: Pathogens that can spread from one person to another

● Common microbial pathogens

The concept and characteristics of viruses

• Viruses are a class of microorganisms that do not have a cellular structure and are composed of nucleic acids and proteins that are inherited and replicated by host cells.
• Features :
  (1) Nanoscale particles, which can only be observed by electron microscope;
  (2) No cell structure , its main components are only nucleic acid and protein;
  (3) Each virus contains only one nucleic acid , either DNA or RNA ;Genome size: 3kb - 1.2Mb
  (4) The virus cannot replicate independently
  , and can only synthesize its own nucleic acid and protein components by using the existing metabolic system in the host living cell; ;
  (6) Under in vitro conditions, it can exist in the state of non-living biological macromolecules and maintain its infective activity for a long time; (7
  ) It is not sensitive to general antibiotics, but sensitive to interferon ;
  (8) Some viruses Nucleic acids can also integrate into the host's genome and induce latent infection .
  

Classification and Morphology of Viruses

● Classification of viruses

• Classification from genetic material : DNA viruses, RNA viruses, protein viruses (eg: prions)
• Classification from virus structure : true virus (Euvirus, virus for short) and subvirus (Subvirus, including viroid, pseudovirus, prion)
• Classification from host type : bacteriophage (bacterial virus), plant virus (such as tobacco mosaic virus), animal virus (such as avian influenza virus, smallpox virus, HIV, etc.)
• Divided by nature : mild virus (HIV), severe virus (rabies virus).

Virus morphology
(1) spherovirus; (2) baculovirus; (3) brick virus; (4) coronavirus; (5) filovirus; (6) streptovirus; (7)
enveloped (8) viruses with spherical heads; (9) insect viruses enclosed in inclusion bodies.

The structure of a virus

(1) Basic structure of virus

• Virus particle ( virus particle): specifically refers to a mature, structurally complete and infectious single virus, mainly composed of internal genetic material and protein shell.
• At its center is the nucleic acid, called the core ( core) or genome, and proteins surround the core, forming the capsid ( capsid). The core and the capsid are collectively called the core shell ( nucleocapsid).
• Capsid functions : ① antigenic; ② protect nucleic acid; ③ mediate the combination of virus and host cells.

(2) Auxiliary structure of the virus

• Some more complex viruses (usually animal viruses, such as influenza viruses) have a lipoprotein bilayer membrane structure outside the viral nucleocapsid, which is obtained when the virus is released by budding and passes through the host cell membrane or nuclear membrane , called the envelope . On the surface of the envelope are virus-encoded glycoproteins inlaid into spike-like protrusions called spikes . Enveloped viruses are sensitive to organic solvents .
• Envelope function : ① Protect nucleocapsid: ② Promote the adsorption of virus and host cells: ③ Antigenic.

● Generally symmetrical : There are only two symmetrical systems of virus particles, namely, helical symmetry (representing tobacco mosaic virus) and icosahedral symmetry (equal axis symmetry, representing adenovirus). Some viruses with more complex structures are essentially the result of the combination of the above two symmetries, so they are called compound symmetry (representing T even-numbered phage)

12.2 Common viruses and related diseases

Introduction to Common Viruses and Related Diseases

• 天花It is a severe infectious disease caused by the smallpox virus . After the patient recovers, there will be pockmarks on the face, so "smallpox" got its name. The smallpox virus reproduces quickly, and it spreads through the air at an astonishing speed. Until the patient's scar is peeled off, smallpox may still be transmitted from the patient to others. Some historians once said that the largest genocide in human history was not achieved by guns, but by smallpox. Thanks to the application and popularization of the vaccinia vaccine, smallpox became the first human infectious disease to be completely eradicated, which can be said to be the pinnacle achievement of human beings in the fight against infectious diseases .

• 流行性感冒(Influenza for short) is a highly contagious and fast-spreading acute respiratory infection caused by influenza virus , mainly through droplets in the air, contact between people or contact with contaminated items.
  Typical clinical symptoms mainly include : sudden onset of high fever, general pain, significant fatigue, and mild respiratory symptoms. Generally, autumn and winter are the high-incidence period, and the complications and death caused by it are very serious.
  Influenza viruses are named for their surface structure (the spike) . For example , H5N1 , H stands for hemagglutinin , its function is to allow the virus to bind to the host cell; N stands for neuraminidase , its function is to release the virus that has replicated itself from the cell . In influenza A, H can be divided into 16 subtypes, and N can be divided into 9 subtypes. All of these subtypes have been isolated from birds.

• 埃博拉病毒First discovered in the Ebola River area of ​​southern Sudan and Congo in 1976, it is a severe infectious disease virus that can cause Ebola hemorrhagic fever in humans and other primates. The Ebola hemorrhagic fever caused by it is The deadliest viral hemorrhagic fever in the world today , with a mortality rate between 50% and 90% , killed 11,000 people in Africa between 2014 and 2016. At present, the Ebola vaccine has been approved for use, which brings hope for overcoming Ebola.

• 艾滋病毒Also known as human immunodeficiency virus ( HIV ), it was first reported by the US Centers for Disease Control and Prevention on June 5, 1981. It mainly attacks the T lymphocytes of the human body, causing immune system dysfunction , and patients often die from infections or malignant tumors of other common pathogens . Although it is generally recognized that "cocktail therapy" can effectively control the progression of AIDS, so far, AIDS is still one of the most difficult medical problems.

• 冠状病毒In terms of systematic classification, it belongs to the order of Nestoviridae-Coronaviridae-Coronaviridae, and it is a large class of RNA viruses that exist widely in nature . The 2019 novel coronavirus (2019-nCoV) is the seventh known coronavirus that can infect humans, and the other six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV Severe Acute Respiratory Syndrome) and MERS-CoV (causing Middle East Respiratory Syndrome).

Retroviral

● Discovery of retroviruses

• RSV (Rou's sarcophagus virus) was discovered in 1910 by Peyton Rous. Peyton Laws extracted substances from chicken tumors and injected them into healthy Plymouth Rock chickens, and found that tumor extracts could induce tumorigenesis.
• Peyton Rous received the 1966 Nobel Prize in Physiology or Medicine.
  

The genome of a retrovirus

• Retrovirus acquires the oncogene src (The virus does not have src originally, and the src is obtained after infecting the host）。
  

Three concepts related to cancer

• 癌基因 (oncogene)Encodes a defective signaling protein . By continually giving cells the signal to divide, they cause tumors to form.Oncogenes are genetically dominant _, possibly encoding defective growth factors, receptors, G proteins, protein kinases, or nuclear regulators of transcription.
  ◆ Proteins encoded by oncogenes :
  

• 抑癌基因 (Tumor suppressor gene)Encodes regulatory proteins that inhibit cell division, repair DNA errors, or regulate apoptosis . When tumor suppressor genes don't work properly, cells can grow out of control, which can lead to cancer. Mutations in these genes haveGenetically recessive , but can lead to tumor formation.
  ◆ Common tumor suppressor genes:
  

• 原癌基因（细胞转化基因） Proto-oncogene ; Cellular oncogene, C-onc
  Existing in the cell genome, it is in a static or low-level (restricted) expression state under normal circumstances, and plays an important role in maintaining the normal function of cells. When it is activated by carcinogenic factors, it will lead to malignant transformation of cells.

● Generation of oncogenes from corresponding proto-oncogenes:

• point mutation
• Chromosomal translocation, in which two genes are fused together to produce a hybrid gene encoding a chimeric protein whose activity differs from that of the parental protein
• A chromosomal translocation that places a growth-regulating gene under the control of another promoter , resulting in abnormal expression of that gene
• Abnormal amplification and replication of a DNA segment containing a proto-oncogene , resulting in overproduction of the encoded protein

Transformation of proto-oncogenes to viral oncogenes

1. The spike of the retrovirus binds to the receptor on the surface of the host cell membrane, endocytosis occurs, and the viral RNA enters the host cell.
2. The retroviral RNA genome is reverse transcribed into double-stranded DNA and integrated into the host cell next to the proto-oncogene
3. After the proto-oncogene is integrated into the viral genome, it is packaged to form a new virus, and the virus is released after a large amount of amplification in the host cell.
4. The new virus continues to infect other host cells, and the proto- oncogene may mutate into an oncogene that causes host cell malignant transformation during the infection process; even if it does not mutate, the expression of the proto-oncogene will increase abnormally .
   

12.2.1 HIV HIV 2× (+)ssRNA

HIV

• HIV is the human immunodeficiency virus. It is this virus that causes acquired immunodeficiency syndrome, or AIDS.
• HIV primarily infects important cells in the human immune system, such as helper T cells (specifically CD4+ T cells ), macrophages , and dendritic cells .
• AIDS is the advanced stage of HIV infection in which a person's immune system is so damaged that it has difficulty fighting off disease and certain cancers.
• Currently, there is no cure for HIV infection.
• HIV is a retrovirus whose RNA genome is reverse transcribed into double-stranded DNA and integrated into the host cell's DNA.
• The HIV core particle contains two copies of its RNA genome , each bound with a primer (host tRNA) and reverse transcriptase (RT). The core particle is surrounded by an envelope containing the spike protein.
• Accessory proteins regulate virion formation and latency .
• Ancient retroviral sequences are present in animal genomes, including human genomes.

HIV genome and structure

• HIV consists of two copies 正链(can be translated directly) composed of single-stranded RNA , which encodes nine genes of the virus (But there are more than 9 expressed proteins , for example, the protein expressed by gag gene will be cut into p17, p24, p7), and is surrounded by a conical capsid consisting of 2,000 copies of the viral protein .p24Caspid
• Single-stranded RNA tightly binds to 核衣壳蛋白, p7and is required for virion development 酶, such as reverse transcriptase, protease, ribonuclease, and integrase.
• A matrixp17 composed of viral proteins surrounds the capsid and ensures the integrity of the virion particle .
• Viral envelope : Two layers of fatty molecules (phospholipids) taken from human
• EnvEncodes a trimeric cap (3 glycoproteins gp120) and a trimeric stem (3 gp41), which together gp160make up the spike .
• The RNA genome consists of at least 7 structural markers (LTR, TAR, RRE, PE, SLIP, CRS and INS) and 9 genes (gag, pol and env, tat, rev, nef, vif, vpr, vpu; sometimes a 10th A gene tev, which is a fusion of tat, env and rev), encodes 19 proteins .
• gag, poland envcontain the information needed to make the structural proteins of new virus particles.

HIV life cycle

1. Following fusion of HIV with the host cell membrane , HIV virions are released into the cytoplasm.
2. The HIV genome is reverse transcribed into double-stranded DNA and integrated into the host genome.
3. DNA is provirally transcribed into retroviral mRNA and exported to the cytoplasm for translation .
4. Retroviral RNA and proteins assemble at the cell membrane and fuse with the cell, releasing the virus outside the cell.
   

★ Key steps in the HIV-1 life cycle

(1) Enter the cell

• ① HIV adsorbs to target cell receptors through the surface glycoproteins of macrophages and CD4+ T cells, then the viral envelope fuses with the cell membrane, and releases the HIV capsid into the cells, thereby entering macrophages and CD4+ T cells;
• ② The process of entering the cell is initiated by the interaction between the trimeric envelope complex ( gp160 spike, spike ) and CD4and chemokine receptors (usually CCR5or , or other interactions). The Gp160 spike contains binding domains for CD4 and chemokine receptors.CXCR4
• ③Combined gp120with integrin α4β7 to activate LFA-1(downstream signaling pathway), which is the main integrin involved in the establishment of viral synapses, which can promote the effective spread of HIV-1 between cells .
• ④ After HIV binds to target cells, inject HIV RNA and various enzymes (including reverse transcriptase, integrase, ribonuclease, and protease) into the cells .
• ⑤ HIV can infect dendritic cells (DC) through the CD4-CCR5 pathway , or the mannose-specific C-type lectin receptor pathway (such as DC-SIGN). DCs are one of the first cells that the virus encounters during sexual transmission. It is thought that DCs play an important role by transmitting HIV to T cells while they capture virus in the mucosa.

(2) Reverse transcription of HIV genome into double-stranded DNA

• ① Shortly after the viral capsid enters the cell, reverse transcriptase releases the single-stranded (+) RNA genome from the attached viral protein and copies it into a complementary DNA (cDNA) molecule.
• ②The reverse transcription process is very error-prone (because reverse transcriptase has low fidelity) and produces mutations that could lead to drug resistance or allow the virus to evade the body's immune system.
• ③ Reverse transcriptase also has ribonuclease activity , which can degrade viral RNA during cDNA synthesis ; it also has DNA-dependent DNA polymerase activity , which is used to generate sense DNA from antisense cDNA .
• ④ The cDNA and its complementary sequence together form the double-stranded DNA of the virus , which is then transported to the nucleus along the microtubules . The viral DNA is integrated into the genome of the host cell by the viral integrase .

● Reverse transcription process

• ① 特定的细胞 tRNAAct as a primer and bind to the primer binding sitePBS of the viral genome ;
• ② Synthesize complementary DNA and bind to the R region (repeated at both ends of the RNA molecule) and U5 (non-coding region) of the viral RNA;
• ③ The domain called RNAse H on reverse transcriptase can degrade RNA 5'末端, thusRemove R and U5 regions；
• ④ Then, the primer "jumps" to the viral genome3'端 , where the newly synthesized DNA strand hybridizes with the complementary R region on the RNA ;
• ⑤ The first strand of complementary DNA (cDNA) is extended ,Most viral RNA is degraded by RNAse H；
• ⑥ After the strand is completed, start the second strand synthesis from the viral RNA ;
• ⑦ Then there is another " jump " where the PBS in the second strand hybridizes with the complementary PBS on the first strand ;
• ⑧ Both chains are further extended and can be integrated into the host genome by integrase.
  

(3) Replication and transcription

• ① This integrated viral DNA may then lie dormant during the latency period of HIV infection . In order to actively produce the virus, certain cellular transcription factors need to be present , the most important of which is NF-κB ( ).NFκBWhen T cells are activated (as other infections occur), secreted NFκBand the virus begins to replicate. This means that the cells most likely to be killed by HIV are those that are currently fighting off the infection .
• ② During virus replication , the integrated DNA provirus is transcribed into mRNA , the mRNA is cut into smaller fragments , these small fragments are exported from the nucleus to the cytoplasm, and then translated into regulatory proteins in the cytoplasm Tat(which can encourage the production of new viruses ) and Rev.
• ③ As the newly produced Rev 蛋白nuclei enter, accumulating in the nucleus , it binds to the viral mRNA and allows unspliced ​​RNA to leave the nucleus , where they would otherwise remain until splicing.
• ④ At this stage, structural proteins Gagand are produced Envfrom full-length mRNA . The full-length RNA is actually the viral genome, which is bound to Gagproteins and packaged into new virus particles.

(4) Virus assembly and release

• ① The final step in the viral cycle is the assembly of new HIV-1 viruses, starting at the plasma membrane of the host cell. EnvThe aggregated protein ( gp160 ) crosses the endoplasmic reticulum and is transported to the Golgi apparatus, where it is cleaved by proteases and processed into the two HIV envelope glycoproteins gp41 and gp120.
• ② The glycoprotein is transported to the plasma membrane of the host cell, where gp41 anchors gp120 to the membrane . Gag(p55)and Gag-Pol(p160)polyproteins bind to the inner surface of the plasma membrane as well as HIV genomic RNA , and virion budding begins from the host cell .
• ③ Maturation may occur in forming buds or in immature virions budding from the host cell. During maturation, HIV proteases cleave polyproteins into individual functional HIV proteins and enzymes.
• ④ The various structural components are then assembled to produce mature HIV virions. This cleavage step can be inhibited by protease inhibitors. The mature virus is then able to infect another cell.

● HIV attenuates the host's immune response

• gp120The protein on the surface of HIV particles falls off and binds to the CD4 receptor on the normal cell membrane , so that the cells are mistaken for virus-infected cells by the immune system and killed (CD4 cells decrease).
• becauseT cell CD4 receptor blocked by gp120, affecting its immune function.
• The gp120 protein of HIV can stimulate the body to produce specific antibodies against the binding site of CD4 and block the function of T cells.
• Cells with viral envelope proteins can fuse with other cells to form multinucleated giant cells without function.

HIV treatment

• HAART( Cocktail Therapy ): Treatment based on protease inhibitors has been available since 1996. Usually two nucleoside analog reverse transcriptase inhibitors ( NARTIor NRTI) plus protease inhibitors or non -nucleoside reverse transcriptase inhibitors . (Inhibits the process of viral reverse transcription, so that the viral RNA cannot be integrated into the genome, cannot be reverse transcribed, but still replicates at a low level. )
• HAART does not cure patients, nor does it uniformly eliminate all symptoms; high levels of HIV-1 virus (often HAART-resistant) relapse if treatment is stopped .
• Research to improve current treatments includes reducing the side effects of current drugs , further simplifying drug regimens to improve adherence, and determining the optimal sequence of regimens to treat drug resistance .
• However, after more than 20 years of research, HIV-1 remains a vaccine target.

12.2.2 Influenza virus (-)ssRNA

flu virus

• Influenza viruses cause periodic respiratory disease pandemics.
• RNA virus,(-)ssRNA
• Subtypes based on specific species and combinations of two proteins (spikes) ( hemagglutinin H1 to H18 and neuraminidase N1 to N10) present on the surface of the virus .
• New virulent strains are produced by recombination of human and avian strains .

● Influenza A virus subtypes circulating in humans

• H1N1 Caused Spanish Flu and 2009 Swine Flu Outbreaks
• H2N2 caused the "Asian flu" in the late 1950s
• In the late 1960s, H3N2 caused the "Hong Kong flu"
• H5N1 spread in the mid-2000s and was considered a global pandemic threat
• H7N7 has unusual zoonotic potential
• H1N2 is currently circulating in humans and pigs
• H9N2, H7N2, H7N3, H5N2 and H10N7.

● Core particle: : RNA, capsid protein, matrix protein, envelope containing spike protein

● Influenza virus life cycle

• Influenza viruses consist of segmented (-) stranded RNA . Each fragment is packaged with a nucleocapsid protein. Fragments from different strains were recombined by co-infection.
• Capsid and matrix proteins block the RNA segment of influenza virus . The matrix is ​​surrounded by an envelope containing the spike protein.
• Envelope proteins mediate viral attachment . The envelope protein includes a fusion peptide that undergoes a conformational change to cause fusion between the viral envelope and the host cell membrane. For influenza, virions are internalized by endocytosis .
• Fusion of lysosomes with endosomes ( endosome) triggers fusion of the viral envelope with endosomes. Then, the viral genome and proteins are released into the cytoplasm. Viral (-) strand RNA fragments are uncoated and enter the nucleus .
• Capped RNA fragments from host mRNA cleavage initiate influenza mRNA synthesis . Viral mRNA returns to the cytoplasm for translation.
• Genomic RNA synthesis is initiated by the nucleocapsid protein ( NP). First, the (+) strand of RNA is synthesized as a template for the (-) RNA strand, which is then packaged in newly synthesized nucleocapsid proteins and exported into the cytoplasm.
• The envelope proteins of influenza viruses are synthesized at the ER and then transported to the cell membrane.
• Influenza virus is assembled on the cell membrane, and the capsid, stroma, and (-) strand RNA components are packaged in the envelope.

12.2.3 SARS-CoV (+)ssRNA

Severe Acute Respiratory Syndrome SARS

● Signs and symptoms

• Initial symptoms are flu-like and may include fever, myalgias, lethargy symptoms, cough, sore throat, and other nonspecific symptoms.
• Fever above 38°C (100°F).
• Shortness of breath may develop later.
• Patients have cold-like symptoms in the first stage, but later it becomes flu-like.

● Severe acute respiratory syndrome coronavirus:SARS-CoV

• Positive strand (+) ssRNAvirus
• Genome: 27-30 kb, 14 ORFs
• Three glycoproteins: S (E2), M (E1) and HE (hemagglutinin esterase)
• S: S1(binds to receptor ACE2) and S2(mediates membrane incorporation )
• Diameter 80-160nm

12.2.4 New Coronary Pneumonia Virus COVID-19 (+)ssRNA

Coronavirus COVID-19

● Currently, the number of infected people has exceeded 163 million, and the cumulative death toll has exceeded 3.37 million.
● Origin unknown.
● Symptoms :

• fever
• cough
• shortness of breath or difficulty breathing
• chill
• trembling trembling
• Muscle pain
• Headache
• sore throat
• new loss of taste or smell

● 序列：Complete Genome Sequence of a 2019 Novel Coronavirus (SARS -CoV- 2) Strain Isolated in Nepal

Coronavirus

● Structure:

• Structural proteins : Spike, Envelope, Membrane, Nucleocapsid protein
• nonstructural protein

● Human coronavirus life cycle diagram
Source: Human Coronavirus: Host-Pathogen Interaction

Application of biological virus

1. Phage (bactericidal) can be used as a specific drug to prevent and treat certain diseases . For example, burn patients apply a dilution of Pseudomonas aeruginosa to the affected area.
2. In cell engineering, certain viruses can be used as a fusion agent for cell fusion (cells have the property of contact inhibition, and fusion will occur in the presence of viruses), such as Sendai virus.
3. In genetic engineering, viruses can be used as carriers of target genes , allowing them to be spliced ​​on the chromosomes of target cells.
4. Viruses added to a specific bacterial culture medium can remove impurities .
5. Viruses can be used as carriers for precision-guided medicines .
6. Viruses can be used as specific insecticides .
7. Viruses also play a key role in the circulation of matter and energy exchange in the biosphere.
8. Viruses can also be used to treat diseases, such as cancer.

12.3 Viral Vectors

• Gene transfer vectors are made from viruses.
• Virulence genes are deleted and efficient promoter sequences from other viruses are inserted .
• To improve the biological safety of the vector, viral vectors have evolved in a form that separates the viral sequences required for packaging and production from the sequences encoding viral proteins .

● Common viral expression vectors

• Lentiviral vector ( Lenti Virus)
• Adenoviral vector ( AV)
• Adeno-associated virus vector (adenovirus associated virus, AAV)
• Reverse transcription vector ( RV)

• Adenoviral vectors circularize and replicate separately from the host genome .
● The retrovirus and lentiviral vectors are integrated in the host genome .
● Safety of gene carrier : Adeno-associated virus : RG1 > Adenovirus : RG1 > Lentivirus : RG2 > Retrovirus : RG3

12.3.1 Lentiviral vector system

• HIV-1Lentiviral vectors derived from human immunodeficiency virus ( );
• For gene expression in mammalian cells (dividing and non-dividing cells) , integration into the genome .
• The HIV-1 genome contains nine open reading frames encoding at least 15 different proteins involved in the infection cycle, including structural and regulatory proteins.
• Several cis-acting elements are present on the genome , including long terminal repeats ( LTRs), TAT activation region ( TAR), splice donor and acceptor sites , packaging and dimerization signals (ψ), Rev response elements ( RRE), and central and The terminal polypurine region ( PPT) is required for gene expression at various stages of the life cycle of different viruses.

● The first generation lentiviral vector

• Vector components (3 plasmids):
  (i) packaging plasmid Packaging plasmid ; (ii) Env plasmid ( envelope plasmid
  ) encoding viral glycoprotein : VSV-G ; (iii) transfer plasmid . Env encoding plasmid
Vector plasmid
• Use genetic engineering to specifically delete the packaging signal (to improve safety) or LTRs of the packaging plasmid to reduce the production of replicative infectious lentiviruses (RCLs).

● The four auxiliary protein coding genes (Vif, Vpu, Vpr and Nef) in the second-generation lentiviral vector

modification system: delete all the accessory genes of HIV in the first-generation lentiviral vector packaging plasmid , and the removal of these accessory genes It does not affect the titer and transfection ability of the virus, and at the same time increases the safety of the vector .

★ Third generation lentiviral vector (currently used)

• Vector components (4 plasmids)
  (i) 2 packaging plasmids , one encoding Revand one encoding Gagand Pol;
  (ii) Env plasmid (envelope plasmid) encoding the viral glycoprotein;
  (iii) transfer plasmid.
• Biosafety is further improved : the production of replicative infectious lentiviruses (RCLs) is reduced
  ① A plasmid that is independent of the vector is constructed by replacing the promoter region of the 5'LTR of the vector plasmid with a strong viral promoter from or ; ② The vector plasmid The deletion of the U3 region sequence of the 3'LTR ensures that the lentiviral particles are self-inactivated after transduction and integration into the genomic DNA of the target cell (U3CMVRSVtat
  No longer has the ability to infect, that is, it can only infect once);
  ③ The number of lentiviral genes required for packaging, replication and transduction is reduced to three (gag, pol, rev);
  ④ Compared with Mulv retroviral vectors, lentiviral vectors are more inclined to integrate into the host genome activity In transcribed regions , the risk of activating proto-oncogene silencing is lower than that of Mulv retroviral vectors.

12.3.2 Adeno-associated viral vectors

• Adeno-associated virus is a class of single-stranded linear DNA-defective virus.
• Genomic DNA is less than 5 kb , without envelope, and the shape is naked icosahedral particles.
• AAV cannot replicate independently , and replication and cytolytic infection are only possible in the presence of a helper virus (eg, adenovirus, herpes simplex virus, vaccinia virus).

● Adeno-associated virus genome

• The two ends of the genome are terminal inverted repeats ( ITR), and the middle genome encodes two proteins: Rep and Cap.
  ITRsPlays a decisive role in virus replication and packaging .
  CapThe protein is the capsid protein of the virus, and the protein is involved in the replication and integration
  Rep of the virus

• AAV can infect a variety of cell types. RepIn the presence of the protein, the viral genome is easily integrated into a specific site on human chromosome 19 : AAVS1loci . This is the only knownfixed-point integrationof mammalian DNA viruses.

• Genome map of wild-type AAV-2
  
  (A) The Rep and Cap genes are flanked by ITRs. Distinct Rep and Cap transcripts are produced by their respective promoters (P5, P19 and P40). Asterisks indicate alternative ACG codons used to produce VP3.
  (B) The secondary structure of AAV-2 ITR, showing RBE(Rep binding element, GAGCGAGCGAGCGCGC; RBE', CTTG) and TRS(terminal resolution site, GTTGG). ITRs are important for AAV DNA replication.

● AAV life cycle

• In the case of adenovirus co-infection , AAV becomes infectious : genome replication, viral gene expression and production of virions.
• In the absence of adenovirus , AAV can establish latency by integrating into chromosome 19 (AAVS1) .
• The latent AAV genome canRestoration of replication by superinfection with adenovirus。
• Both phases of the AAV life cycle are regulated by complex interactions between the AAV genome and AAV, adenovirus and host proteins.

Adeno-associated virus vector

a. Schematic diagram of AAV. The single-stranded genome is surrounded by a protein capsid.
b. DNA genome of AAV. The coding rep, capand aapthe open reading frame of the 4.7 kb genome are terminated by ITRs.
c. Components of AAV vectors.
① Insert the therapeutic transgene and associated promoter and polyadenylation sequence (polyA) between the viral ITRs.
② Vector expresses AAV rep and cap
③ Vector expresses adenovirus E2A, E4 and VA RNA
abbreviations: AAP, assembly activator protein; AAV, adeno-associated virus; ITR, inverted terminal repeat; PolyA, polyadenylation sequence; VA, virus relevant.

● Advantages of Adeno-Associated Viral Vectors

• Adeno-associated virus vector AAV Helper-Free System, a packaging system that does not require adenovirus helper vectors.
• Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus, due to its good safety and wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo . It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide.

● Disadvantages of adeno-associated viral vectors

• The AAV vector has a small capacity and can only accommodate up to 4.7kb foreign DNA fragments at present;
• Wild-type AAV is infective in 40% - 80% of adults and is not conducive to vaccine production; it may cause immune rejection in the presence of adenovirus .

● Genes suitable for AAV expression

1. The length of the therapeutic gene should not be too large. The total capacity of AAV is 4.7kb, andIncluding AAV's own ITR, promoter region and RNA tailing signal；
2. The gene needs to be continuously expressed , and the protein can be secreted or non-secreted;
3. Long-term expression of the target gene without toxic side effects;
4. Immediate expression of the target gene is not required;
5. High level expression of genes is not required (Virus-based expression vectors, the expression level will be lower）；
6. AAV vectors have high transduction efficiency for target cells.

12.3.3 Herpes simplex virus vectors

Herpes simplex virus HSV : DNA virus

• Herpes simplex virus is a virus that causes recurring sores on the mucous membranes of the mouth or genitals. Initial transmission is through oral or genital contact, followed by a latent, reactivated viral outbreak in ganglion neurons .
• Herpes virions contain a double-stranded DNA genome packaged in an icosahedral capsid. The capsid is surrounded by many matrix proteins and an envelope.
• HSV attachment may involve multiple receptors.
• Microtubule scaffolds transport herpes virions to the nucleus and insert into the DNA genome . HSV assembly occurs at the nuclear envelope or other membranes. Virions are released from cells by exocytosis, and rapid release leads to mucosal pathology.
• The DNA genome of HSV was synthesized by the rounding method (high replication efficiency), using a viral DNA polymerase supplemented with viral and host-produced components.
• Expression of infectious mRNA results in the production of capsid, matrix and envelope proteins, thereby HSV assembly.
• LATExpression of the protein often leads to latent infection in nerve cells where DNA silencing persists for months or years.

Herpes simplex virus vector

● The amplicon system includes:

1. Amplicon plasmid containing Orithe herpes simplex virus (HSV) origin of DNA replication ( ), HSV packaging sequence ( Pac), antibiotic resistance gene ( Amp) and promoter-transgene cassette ;
2. Helper virus to provide virus replication function.

● The helper virus genome with the packaging signal deleted is cloned into a bacterial artificial chromosome ( BAC).
● When eukaryotic cells are co-transfected with amplicon and HSV-BAC DNA , the resulting virus particles can only recognize and package the amplicon plasmid, resulting in a helper virus-free amplicon stock .

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Assignment : Find the nucleic acid sequence of the S protein of SARS-CoV2 (secreted outside the cell), and construct it into a eukaryotic expression vector to allow it to be secreted and expressed outside the cell. It is required to design primers and describe the design scheme.
Secreted to the outside of the cell : a secretory peptide is fused to the S protein, placed behind the secretory peptide, and in the same frame.

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