BIOL 4190 Final
Virion characteristics of the familyPapillomaviridae
*important! Small dsDNA viruses that infect mammals and birds. § Over 100 human papillomavirus (HPV) types found, differentiated by their DNA sequences. § Enter the body through small abrasions and infect keratin-making cells (keratinocytes). § Each HPV type infects a preferred site, such as the hands or the genitals, and infection may result in a benign wart (papilloma) or a carcinoma.
Effect of HAART on the concentration of HIV-1 in the blood
-HAART (highly active antiretroviral therapy) does not elimate HIV infection from the body -HAART can markedly reduce death rates from AIDS and treatment of HIV-infected women has significantly reduced risk of mother-to-child transmission -drug resistant strains of HIV may emerge. If patients lapse from their routine taking durgs
HBV Genome Structure
-Partly single-stranded and partly double-stranded, with a short triple-stranded sequence. -3.2 kb in length -The 5 ends of DNA strands are covalently linked to a capped RNA (on the short strand) or a protein (P) (on the long strand)
Describe the four major theories that explain why viral infection can lead to the development of cancer.
1. Deliberate' Interference with Control of the Cell Cycle - Virus produced proteins inhibit the function/stability of important cellular proteins involved in cell cycle control 2.Accidental' Activation of Cell Genes-Retrovirus produced proteins or provirus integration can enhance the expression of host proto-oncogenes 3. Oncogenes Carried by Virus-Some retroviral genomes carry oncogenes derived from cell proto-oncogenes which can become oncogenes when mutated or aberrantly expressed. 4. Damage to Immune Defenses-Interactions between cell proteins and proteins produced by oncogenic viruses can lead to breakdown of immune defenses that may allow the development of a cancer
Genetic Organization of Phage ϕX174
11 proteins encoded altogether. The first entire DNA genome to be sequenced. Proteins are generated through extensive use of overlapping genes translated in alternative reading frames or employing different start codons. All genes are transcribed in the same direction.
Why was the 1918 Spanish Flu So Deadly?
1918 was an avian—not swine strain.
About ____ of human cancers associated with viruses
20%
HBV Transcripts
4 types of mRNA transcribed: 3.5 kb (to be used as mRNA or pregenome) 2.4 kb 2.1 kb 0.9kb
5.Gene expression in ssRNA phages is often regulated by secondary structure of the viral genome. Please give detailed evidence that supports this notion.
A complex secondary structure controls gene expression via differential access of the ribosome binding sites (RBSs) to host ribosomes during translation.
Hepatocellular Carcinoma (HCC)
A fatal disease with very poor response to current chemotherapy HBV DNA sequences are found in HCC tumor DNA. Integration of HBV DNA occurs in breaks in the cellular DNA of hepatocytes (liver cells). Chronic HBV infections cause ongoing inflammatory responses and oxidative damage to chromosomal DNA of hepatocytes.
Superinfection immunity:
A resident prophage protect the host from superinfection by the same or similar strains of phages by repressing the incoming phage genome.
Geographical Prevalence of Hepatitis B Virus (HBV)
About a third of the world's population, more than 2 billion people, have been infected with the hepatitis B virus. This includes 350 million chronic carriers of the virus.
Why is Influenza More Prevalent During the Winter?
Aerosol spread of influenza virus is dependent upon humidity and temperature. The virus was best transmitted at low humidity (20%) and colder temperatures (5oC or 41oF). Supporting evidence that weather conditions play a role in influenza virus transmission.
Treatment of HBV infection
Alpha-interferon has been used for some years to treat HBV-infected persons. This treatment does not eliminate the infection, but it results in a significant reduction in viraemia in about 20-30 per cent of cases. Side-effects may occur, such as influenza-like symptoms and weight loss, which may necessitate reducing the dosage or even discontinuing the treatment. The drug lamivudine, a nucleoside analogue, is also used to treat HBV infection, suppressing virus replication with a low incidence of side-effects, administered by mouth rather than by injection and cheaper. Longterm treatment, however, results in the appearance of lamivudine-resistant HBV mutants, though they appear to be susceptible to other nucleoside analogues such as adefovir. Immune globulin used as an adjunct to HBV vaccine in preventing HBV transmission from an infected mother to fetus
The 1918 Influenza Pandemic
Also called the Spanish flu Killed 675,000 Americans, decreasing the life expectancy in US by 11 years National average death rate was 4.39 out of every 1000 people Death rates peaked in October of 1918 Unique epidemic: healthy adults ages 20-40 died of the flu, in addition to children and elderly (who are usually high-risk)
Lamivudine/3TC
An analogue of cytidine. Inhibits reverse transcriptases, including those of HIV and hepatitis B virus (HBV). Used, usually in combination with other therapies, to treat infections with HIV or HBV. Does not eliminate the infections, but it can control them. Often used in combination with α-interferon for treatment of HBV infection.
Aciclovir/acyclovir
Analogue of guanosine. Function: inhibition of herpesvirus DNA synthesis. Very safe drug, with almost no side-effects. Widely used for the treatment and prevention of diseases caused by herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV).
Azidothymidine (AZT)
Analogue of thymidine. Also known as zidovudine. It had been investigated as a possible anti-cancer drug and it was found to inhibit the reverse transcriptase of HIV. Becomes phosphorylated to the 5 triphosphate after uptake into a cell. AZT triphosphate binds more strongly to the viral reverse transcriptase than to the cell DNA polymerase, and the reverse transcriptase binds AZT triphosphate in preference to deoxythymidine triphosphate. Also interfere with cell DNA synthesis and, like ganciclovir, it can damage the bone marrow, causing severe side-effects when large doses are used.
7.Describe the difference between antigenic drift and antigenic shift.
Antigenic drift: gradual accumulation of genetic mutations in viral genome. Antigenic shift: a result of genome re‐assortment
Cancer Today
At least six viruses are thought to contribute to human cancers: Hepatitis B virus Hepatitis C virus Human Papillomavirus Epstein-Barr virus Kaposi's sarcoma-associated herpesvirus Human T-lymphotropic virus types I and I Approximately 20 per cent of cancer cases in females and about eight per cent in males are thought to be caused by viruses
Resistance to Bacteriophage Infection
Bacteria become resistant to phage infection when the cell receptors are altered by mutation There is interest in engineering new receptor-recognition elements into the tail fibers of well-characterized bacteriophages so they can infect genetically distant hosts
6.Which HBV protein can not be found in the non-infectious HBV particles?
C and P proteins
HIV Fusion Inhibitors
Can inhibit fusion between the membranes of an HIV-1 virion and a potential host cell by binding to gp41 and inhibiting the conformational change. Example: enfuvirtide (INN or FUZEON), a 36-amino-acid peptide with the sequence of a gp41 region that interacts with another gp41 sequence to cause the conformational change. Enfuvirtide prevents infection of a cell with HIV-1 but has much lower activity against HIV-2, which has a trans-membrane glycoprotein only distantly related to that of HIV-1. A disadvantage of enfuvirtide: it has to be given by subcutaneous injection, whereas the other anti-HIV drugs can be given orally.
Papillomavirus-linked cancers
Carcinoma: a type of malignant and invasive epithelial tumor that spreads by metastasis and often recurs after excision. Cervical carcinoma: the third most common cancer in women, with approximately half a million new cases and 280 000 deaths in the world each year. Most of these cancers result from infection with a papillomavirus.
Other Approaches for Developing Live Attenuated Virus Vaccines
Cold-adapted' virus strains. Examples, influenza and respiratory syncytial virus vaccines (not sufficiently attenuated for use in children). Reassortants. Example, Rotavirus reassortants with some genes derived from a human virus and some from an animal virus. Reverse genetics. Example, a respiratory syncytial virus vaccine created by introducing mutations into viral genome.
Low pH-dependent Fusion of Influenza A
Conformational changes in influenza H protein trigger membrane fusion. (due to pH?)
Indicate whether the statement :"The rapid eradication of smallpox disease can be attributed to the fact that smallpox virus infection often produces clear disease symptom" is correct.
Correct. High‐incidence clear disease symptom helps us to monitor virus spread, which is important for us to eradicate the virus.
Rational Design of Anti-viral Drugs
Decide a target step of viral replication. Choose a target protein involved in that step. Determine the three-dimensional structure of the target protein and select a target site for potential drugs. Use computer programs to design compounds that will bind to the target site with the aim of inhibiting the activity of the virus protein. Lab test. Clinic trials.
Nevirapine: Non-nucleoside Inhibitor of Reverse Transcription
Developed from rational design. Targets different site in the reverse transcriptase to those targeted by the nucleoside analogues.
Ritonavir: HIV Protease Inhibitor
Developed from rational design. Can fit into the active site of the HIV protease.
Transcription of Viral RNA
Differences between influenza virion (-) genome RNA, viral (+) mRNA containing the snatched cellular mRNA cap structure, and (+) viral complementary RNA or template RNA that is a complete copy of the genome RNA segment. influenza viruses use their own RNA depedent Rna polymerase of genome replication in the nucelus
Maturation of HIV-1 Virions
Dimers formed by Gag-Pol proteins undergo selfcleavage to form the virus enzymes, including the protease. The protease then cleaves the Gag polyproteins into the constituents of the mature virion.
Michael J. Bishop and Harold E. Varmus
Discovered that the src gene of RSV is found in the normal DNA of chickens. Their work demonstrated that oncogenes are cellular genes that were hijacked by viruses from cells.
Cellular Pathogenesis
Droplet transmission Virus enters respiratory tract Attaches to ciliated columnar epithelial cells lining the sinuses and airways
Drug Resistance
Emerged as a result of natural selection. Genotypes encoding drug resistance can arise rapidly during high mutation rate of viral replicases. Drug resistance of viruses is relative, rather than absolute. Drug-resistant virus isolates are found to have one or more mutations in the genes encoding the proteins that are the drug targets. Alternative drugs are used when drug resistant strain emerges. Use of a combination of drugs with different targets can prevent drug resistant strain to emerge (highly active antiretroviral therapy (HAART)).
Influenza A Virus Particle
Enveloped Contains hemagglutinin (H) antigen glycoprotein spikes on the surface and neuraminidase (N) antigen spikes. M2 ion channel protein 8 segments of ssRNA of negative polarity
1.Describe the structure feature of influenza viruses (hint: envelop, segmented genome, genome constitution, matrix protein, nucleocpasid symmetry etc.)
Enveloped virus with segmented genome, which is minus‐stranded. Has matrix protein. Viral genome packaged by nuclear protein in helical symmetry
Expression of A gene of ssRNA phage
Expression of the A gene occurs independently and is limited to periods of nascent plus strand synthesis, during which the initiator site of A becomes exposed transiently when plus strand synthesis begins. Thus, the number of molecules of A protein is maintained in line with the number of new RNA plus strands.
Structure of ssDNA Phage ϕX174
F protein: forms the main shell. G protein: forms the spikes that protrudes on the icosahedral five -fold axes. H protein: A molecule of H can be found on each spike and acts as a pilot protein. H proteins span the capsid through the channels formed by the G proteins. The projections are involved in attachment to the host and delivery of the genome into the host cell. J protein: Highly positively charged DNA binding protein, associated with the ssDNA genome
Antivirals for influenza treatment
First drugs: M2 inhibitors (prevent uncoating step) -Amantidine (sold as Symmetrel) -Rimantidine (sold as Flumadine) New class of antivirals: N inhibitors (prevents neuraminidase from cleaving sialic acid during budding) causes viruses to clump at the cell surface, reducing viral spread -Oseltamivir (sold as Tamiflu, pill form) -Zanamivir (sold as Relenza, must be inhaled)
________ protein Must be cleaved by cellular proteases for the virus to be infectious.
H protein
Adult T Cell Leukaemia and Human T-Lymphotropic Virus 1
HTLV-1 is a retrovirus and the tumor cells each have a copy of the proviral DNA integrated into a chromosome. Regions with a high prevalence of HTLV-1 also have a high prevalence of these tumors.
Screening Compounds for Anti-viral Activity
Has shown to be successful in identifying antiviral drugs. Some of the compounds chosen in the past had already been shown to have anti-tumour-cell activity. Involves testing dilutions of the compounds against a range of viruses growing in cell cultures. Inhibition of cytopathic effect (CPE) or of plaque formation are often recorded as evidence of antiviral activity. Has been replaced by the rational design of drugs in many cases.
Hepatocellular Carcinoma and Hepatitis Viruses
Hepatocellular carcinoma accounts for 4-5% of cancer cases in the world. Both HBV and HCV are found to be associated with hepatocellular carcinoma. The HBV DNA is integrated into the cell genome in most of the tumors. Virus DNA often undergoes rearrangements with only X protein produced in tumor. HCV is the only class IV virus (plus-strand RNA virus) that is known to be oncogenic. Its genome is not found routinely in the cancer cells, in contrast to the other human oncogenic viruses.
6.Transcription of Influenza A virus mRNAs needs primers. Where do the primers come from?
Host primary mRNAs
Hepadnaviruses
Hosts: humans and other primates Birds (e.g. wood duck) Virion: Enveloped 40-48 nm diameter Icosahedral capsid Genome: DNA (partly single-stranded) 3 kb --Very small genomes used with great economy to encode the virus proteins and to control expression of the virus genes. --Replicated via an RNA intermediate. Thus also called pararetroviruses alongside with some other plant DNA viruses that replicate via an RNA intermediate (CaMV)
What's the definition of IC50 and SI in antiviral drug study? An ideal antiviral drug should have what kind of IC50 and SI values?
IC50 and SI vary depending on the virus strain and the cell type. An ideal antiviral drug should have low IC50 value but high SI value.
Tumors develop from transformed Cells
Ideal model used to test tumorigenicity: a) lack a thymus, thus are highly immunocompromised, and relatively receptive to engrafted cells from genetically unrelated sources. b) hairless, therefore it is easy to monitor closely the progress of tumor formation after transformed cells have been injected under the skin
Lysogenic (Temperate) Infections
Infection of the hosts does not lead to killing of the hosts. Genome becomes integrated into a specific region of the host chromosome The integrated bacteriophage genome is called a prophage The viral DNA replicates every time the cell copies its chromosomal DNA during cell division
Translational Control Mechanisms
Influenza A suppresses the interferon system in host cells
DNA Vaccines
Introduction into the vaccinee of DNA encoding an antigen, with the aim of inducing cells of the vaccinee to synthesize the antigen. Advantage: Steady supply of new antigen to stimulate the immune system, as with live virus vaccines. Because the antigen is produced within the cells of the vaccinee, it is likely to stimulate efficient T-cell mediated responses. Examples, HIV-1, SARS coronavirus, West Nile virus and foot and mouth disease virus. Trials have been carried out in mice, pigs, horses and humans.
1.Which HBV protein contains the virus attachment site?
L protein
Types of Virus Vaccines
Live attenuated virus vaccines They infect but do not cause disease *not safe for immunocompromised people!, trigger T cell immunity, risk of disease Inactivated viruses They cannot infect but expose the person to the viral antigens safe, does not trigger T cell mediated immunity, no risk of disease Subunit vaccines They contain only a subset of viral proteins safe, does not trigger T cell mediated immunity Recombined virus vaccine Harmless recombined virus that produce antigen from virulent virus triggers both B and T cell mediated immunity Nucleic acid vaccine DNA or mRNA that produces virus antigens after injection high potency, rapid development
Bacteriophages Create Pathogenic Bacteria in Nature
Lysogenic conversion—occurs when a bacteriophage alters the phenotype of a lysogenic bacterium e.g. Corynebacterium diphtheriae produces a toxin responsible for diphtheria only if it carries the temperate bacteriophage called . The tox gene is located near one end of the phage genome
Types of Bacteriphage Infection
Lytic Infection and Lysogenic Infection
Influenza A Uncoating Step
M2 ion channel in the viral envelop allows H+ ions to penetrate the virion, weakening the viral M1 matrix protein from the viral RNA, NP, and transcriptase complex (RNP) RNPs released into the cytoplasm Amantidine (sold as Symmetrel) and rimantidine (sold as Flumadine) block the M2 ion channel function, interfering with uncoating.
Attachment of MS2 F pili
MS2 infection involves attachment of virion to pilin (the pilus subunit) along the length of a sex (F) pilus of a susceptible host via the A protein. Such binding of phages can block conjugation in E. coli. The plus strand RNA genome directs protein synthesis immediately upon infection.
Theory II: 'Accidental' Activation of Cell Genes
Major supportive evidence: Retrovirus produced proteins or provirus integration can enhance the expression of host proto-oncogenes
Theory I: 'Deliberate' Interference with Control of the Cell Cycle
Major supportive evidence: Virus produced proteins inhibit the function/stability of important cellular proteins involved in cell cycle control
Genetic maps of ssRNA Phage Qβ
Maturation (A2) protein also mediates cell lysis. Minor coat protein (A1) is generated as a result of reading through the coat protein gene into the intergenic region, due to a leaky coat protein terminator (UGA). The readthrough protein constitutes 3 to 7% of the virion protein and has a role in host infection. The ssRNA genome not only encode the phage proteins but also determines specific secondary and tertiary structure that regulates translation, replication and other functions.
Risks in Using DNA Vaccines
May trigger an anti-DNA autoimmune disease. May create cancer-causing mutations when injected DNA is inserted into host genomes.
General Features of Bacteriophages
Morphologically and genetically diverse virus species that infect bacteria Important factor control bacterial abundance and distribution Can be broadly classified as virulent or temperate. Temperate phages have alternative replication cycles: a productive, lytic infection or a reductive infection, in which the phage remains latent in the host, establishing lysogeny.
Vaccines
Most effective way to prevent influenza Flu vaccination time in the U.S. October and November Vaccine grown in eggs Inactivated trivalent vaccine, a cocktail of 3 virus strains 2 strains of Influenza A 1 Influenza B strain Live, attenuated vaccine, LAIV Licensed in 2003 Only approved for healthy people ages 5-49 years
How Do Viruses Cause Cancer?
Most oncogenic viruses can have persistent infection. But persistent infection does not necessarily causes cancer. Thus other factors must be involved, which include: state of the host environmental factors the cellular changes induced by the virus infection.
Describe the basic biological properties, such as capsid symmetry and genome constitution, of human papillomavirus
Naked virus with icosahedral capsid. The genome is circular dsDNA
Influenza Virus Neuraminidase Inhibitors
Neuraminidase plays a vital role during the final stages of virion budding from infected cells. Therefore when inhibited the virions are not released. Each neuraminidase spike on the virion surface is made up of four monomers, each consisting of a 'balloon' on a 'stick'. The monomer has a deep cleft that forms the active site of the enzyme. Compounds bound in the cleft can inhibit neuraminidase activity. Example, oseltamivir (Tamiflu).
HBV Vaccine Recommendations by the CDC
Newborns should receive a birth dose before leaving the hospital Children age 19 and under should receive the HBV vaccine series. HBV vaccine is the first vaccine to prevent a cancer. 1990-2002 incidence of hepatitis B-related liver cancer decreased 67%
Why Smallpox Can Be So Easily Eradicated?
No animal reservoir Lifelong immunity(no antigenic shift or drift) Subclinical cases rare Infectivity does not precede symptoms One serotype (DNA virus that evolves slow) Effective vaccine Major commitment by governments to surveillance and containment
Is it appropriate to inoculate immunecompromised individuals with live attenuated virus vaccine?
No. Live attenuated virus vaccine can cause diseases in immunocompromised individuals
Does inactivated virus vaccines trigger both T cell and B cell mediated immune response?
No. They only trigger B cell mediated immune response
EM Image of Hepatitis B Viruses
Non-infectious particles may function as decoy for antibodies, thereby providing virions with some protection from the host immune system
Indicate whether the statement "Oncogenic virus usually refers to those viruses that can replicate in cancer cells" is correct.
Not correct. Oncogenic viruses are those viruses that are able to cause cancer.
Risks in Using Live Attenuated Vaccines
Nucleotide substitutions during virus replication might result in reversion to virulence. Recombinants between vaccine strains and wild type strains may form.
Lysogenic infection:
Often refers to a type of bacteriophage infection that ends up with viral genome being integrated into host genome as prophage or being copied along with host genome. Lysogeny: A status of lysogenic infection.
Lytic infection:
Often refers to a type of bacteriophage infection which culminates in host cell destruction.
HBV pregenome
P binds at a site within a sequence known as ε (Epsilon) that has a high degree of secondary structure and can be found at both ends of the pregenome RNA. But P only binds the 5 end. The ε structure with its bound P protein acts as the packaging signal for incorporation of the pregenome RNA into a capsid.
Complete Replication Cycle of ssRNA Phages
Phage assembly involves spontaneous aggregation, in which the (+) RNA associates with the A protein and is then encapsidated through specific recognition of the phage RNA by coat protein dimers.
Uses of Aciclovir in Clinic
Prevention of cold sores Prevention and treatment of genital herpes Treatment of HSV encephalitis (early treatment reduces the mortality rate) Protection of immunocompromised patients after exposure to VZV Treatment of shingles (pain can be reduced significantly).
Viral Replication
Properties of the Influenza A Particle Immediately after isolation, particles are filamentous (left panel) After several passages in cell cultures, the particles become spherical (right panel)
What kind of genes are defined as proto-oncogene?
Proto‐oncogenes are cellular genes that promote the normal growth and division of cells. They can cause cancer when overexpressed.
What kind of genes are proto-oncogenes?
Proto‐oncogenes are cellular genes that promote the normal growth and division of cells. They can cause cancer when overexpressed.
2.Which enzyme replicates the genome of influenza viruses ?
RNA‐dependent RNA polymerase, which is formed by three viral proteins, PA, PB1 and PB2
Tumor suppressor Rb
Rb (retinoblastoma protein) binds to transcription factor E2F and prevents gene expression of proteins needed to go to S phase.
Uncoupling of Genome Replication and Protein Translation
Replicase may block the coat protein ribosome entry site to suppress the translation of coat protein. Coat protein translation can resume when sufficient (−) RNA has been synthesized. The replicase associates with a number of host proteins to form a phage RNA-specific polymerase, which makes both (+) and (−) strands of RNA.
Characteristics of ssRNA phage Genome Replication
Replication occurs in two stages and involves replicative intermediates (RIs) composed of multiple, nascent RNA strands synthesized from a single template, and replicase Secondary structure, which forms as each new strand is synthesized from the template strand, serves to keep the two strands apart, in turn preventing formation of a double-stranded intermediate. Replicase is error prone and the lack of a double-stranded intermediate means there is no template for error correction.
Virion Subunit Vaccines
Require that large amounts being injected and it may be difficult to obtain an inflammatory response required for a vigorous response without overdoing it. Usually purified components of virions. Example, the haemagglutinin (H) and neuraminidase (N) surface glycoproteins in the case of influenza the vaccines. Cause fewer side-effects, as compared to the inactivated vaccines, but induce poorer immune responses, so multiple doses are necessary to provide adequate immunity.
Antigenic Shift
Responsible for Pandemic Strains Antigenic shift occurs when the influenza A virus acquires a new H or N gene through genome re-assortment.
Antigenic Drift
Responsible for seasonal influenza strains Antigenic drift is the gradual accumulation of new epitopes on the H protein (and, to a lesser degree, the N protein).
Viruses also Cause Animal Cancers
Retroviruses, such as Rous sarcoma virus, cause cancer in chicken, cats and Cattle. A type of herpesvirus causes Marek's disease in chickens, which is a lymphoma. Papillomaviruses cause cancers in cattle, horses and rabbits.
The HBV virion
Roughly spherical, 42 nm diameter S: small envelope protein M: medium envelope protein L: large envelope protein P: polymerase (one molecule is covalently linked to the 5 end of the (-) DNA The virion may contain a second molecule of P
Safety of Anti-viral drugs
SI: selectivity index IC50: 50 percent inhibitory concentration An ideal antiviral drug should have low IC50 value but high SI value IC50 and SI vary depending on the virus strain and the cell type.
Some oncogenic viruses can produce proteins that are able to inhibit the function of P53(a tumor suppressor). Give 4 examples of this type of viral proteins.
SV40 large T (= tumour) antigen (binds to p53 and Rb). KSHV latency-associated nuclear antigen (binds to p53 and Rb). HTLV-1 Tax protein (binds to p53). HBV X protein (binds to p53)
Strategies for the Development of Anti-viral Drugs
Screening compounds for anti-viral activity Rational design of anti-viral drugs
Single-stranded RNA Phages
Small, icosahedral viruses of the family Leviviridae ( levis =light). High mutation rates during replication, some of the smallest RNA genomes known. Plus strand viruses (with the genome acting as mRNA), containing only a few genes, and infect various Gram-negative bacteria, including E. coli, Pseudomonas spp. and Caulobacter spp.
Theory III: Oncogenes Carried by Virus
Some retroviral genomes carry oncogenes derived from cell proto-oncogenes which can become oncogenes when mutated or aberrantly expressed. Nearly all of the oncogene-carrying retroviruses are defective as a result of genome deletions. These viruses can replicate only with the help of an endogenous retrovirus, or in a cell co-infected with a helper virus. The oncogene-carrying retroviruses have the ability to induce rapid formation of tumors (1-6 weeks post-infection).
Lysogenic (Temperate) Infections, cont
Some temperate phages encode transposase which allows the bacteriophage to insert randomly into the chromosome Other bacteriophages integrate into site specific locations within the chromosome All bacteriophage gene expression is repressed by a repressor protein, which confers superinfection immunity.
HBV structures present in patient's blood
Spherical 17-25 nm particle, most abundant HBV particle in carriers Filamentous particles, less numerous, up to 200 nm in length, noninfectious Dane particles, infectious, 42 nm in diameter, enveloped, least abundant, containing the viral DNA polymerase (RT), protein kinase C, heat shock 90 protein associated with the viral genome.
Replication cycle of Ff phages
Stage 1: generation of the double-stranded replication format (RF) •Host RNA and DNA polymerases are involved in the (-) DNA synthesis. • •The minus strand can be transcribed and translated into phage proteins, such as gene II protein (pII), an endonuclease, which is required for the second stage of replication.
Replication of F-specific filamentous phage DNA
Stage 2: Multiplication of RF; rolling circle replication Stage 3: Amplification of ssDNA; RF → ssDNA pV accumulates and serves as a translational repressor of pII, in turn regulating DNA synthesis. The nucleoprotein complex of pV dimers and ssDNA forms a filamentous structure which is targeted to the cell membrane for virion assembly.
Mechanism of Aciclovir
Strongly inhibits virus DNA synthesis but has very little effect on cell DNA synthesis. The first phosphorylation of aciclovir is carried out by the virus thymidine kinase (TK). The cell TK is produced only at certain stages of the cell cycle and it has a much lower affinity for aciclovir than the viral enzyme, so there is very little phosphorylation of aciclovir in uninfected cells. Aciclovir triphosphate competes with deoxyguanosine triphosphate (dGTP) for incorporation into the new strand during DNA synthesis.
Theory VI: Damage to Immune Defenses
Supportive evidence: Interactions between cell proteins and proteins produced by oncogenic viruses can lead to breakdown of immune defenses that may allow the development of a cancer: -Papillomavirus proteins interfere with apoptosis, thereby preventing the death of virus-infected cells. -Some of the proteins produced by EBV and KSHV in latently infected cells can interfere with acquired immune responses.
Anti-viral Drugs: Nucleoside Analogues
Synthetic compounds structurally similar to nucleosides. Act by interfering with the synthesis of virus nucleic acids. Become phosphorylated at the 5 carbon (or its equivalent) to become a nucleotide analogue after being taken into a cell. The 5 triphosphate derivative of the nucleoside analogue is the active form of the drug and acts as a competitive inhibitor of a viral replicase. When incorporated into a growing strand, the nucleic acid synthesis will be terminated, as that found in the dideoxy method of DNA sequencing (the structure of the nucleotide analogue prevents it from accepting the next nucleotide).
6.Phage T4 was suggested to be used as therapeutic agent for the treatment of diarrhea caused by some E. coli. strains. Why?
T4 is a virulent phage which always cause lytic infection
Nucleocapsid Assembly
The C protein forms dimers, which are assembled into capsids. The C protein can self-assemble. A molecule of P protein, along with several cell proteins, binds to a molecule of 3.5 kb RNA, which will function as pregenome RNA.
Virion structure F-specific filamentous phage
The capsid is a flexible protein filament of variable length, depending on genome size. Major coat protein arranged in an overlapping array to form a helix. The minor coat proteins are present at the ends of the filament. The single-stranded circular supercoiled genome is housed lengthwise in the centre of the filament, orientated so that the hairpin packaging loop is located at the pVII/pIX end.
HIV Attachment and Entry
The interaction of gp120 with the receptor and co-receptor results in a dramatic re-arrangement (conformational change) of gp41. This change will then proceed to fuse the membranes of the virion and the cell membrane for the release of virion content into cytoplasm.
Genetic Maps of ssRNA Phage MS2
The lysis protein is produced when ribosomes traversing the coat protein gene fail to maintain the correct reading frame such that once translation of coat protein terminates they can reinitiate at the start of the lysis protein ORF, by shuffling a short distance. Efficiency of translation of the lysis protein ORF by such reinitiation is low. Thus only small amounts of lysis protein are synthesized, in turn guaranteeing that sufficient copies of coat protein will be available for assembly of virus particles before cell lysis occurs.
Prevention of HBV infection
The original HBV vaccines consisted of non-infectious spheres and filaments extracted from blood donations from HBV carriers. Now much vaccine is produced using recombinant yeast cells containing the gene for the S protein. Vaccination programs have been successful in reducing the percentage of HBV carriers. The requirements to mass produce HBV vaccine cheaply and to vaccinate many more of those at risk are major challenges facing the world.
Cloning vectors
The relatively small size and non-lytic infection cycle suggest easy manipulation and straightforward usage as cloning vectors. Since filament size is governed by size of the genomic DNA, insert DNA can be very big. Vectors are based on the RF, which is easily obtained from infected cells. Cloned ssDNA can be recovered from the phage particles for direct use in DNA sequencing, designing DNA probes and site-directed mutagenesis.
7.Filamentous ssDNA phages have been extensively used as cloning vectors. Why?
The relatively small size and non‐lytic infection cycle suggest easy manipulation and straightforward usage as cloning vectors. Since filament size is governed by size of the genomic DNA, insert DNA can be very big. Vectors are based on the RF, which is easily obtained from infected cells. Cloned ssDNA can be recovered from the phage particles for direct use in DNA sequencing, designing DNA probes and site‐directed mutagenesis.
Viral mRNAs are not Cannibalized for Their 5' Caps
The viral PB2 polymerase proteins selectively "snatch" caps. Binds to a specific sequence that is complementary to nucleotides 1-12 of the 3' ends of each vRNA segment.
What are tumor suppressor genes?
They are genes that suppress or inhibit the conversion of a normal cell into a cancer cell. These genes cause cancer when turned off.
Not all types of cell transformation lead to tumorigenesis
Transformation that may lead to tumorigenesis often shows the following characteristics: Loss of anchorage dependence Loss of contact inhibition Decreased requirements for growth factors
Bacteriophage translation
Translation may start before transcription is complete All ORFs within an mRNA are translated and several may be translated concurrently
Translation, Transcription and Virion Assembly of Phage ϕ6
Translation of the L segment produces the early proteins that assemble to form the polymerase complex; the M segment produces structural proteins for membranes and spikes in particular, and the S segment produces structural proteins for the capsid, membrane assembly, lysis and entry, and a non-structural protein for envelopment of the capsid. Transcription of the genome is temporally controlled and virions assemble in the cytoplasm, with their envelope deriving from the host. Packaging of the plus strands occurs in the order S-M-L. These single-stranded precursors are then replicated into mature double-stranded genomes inside the capsid (conservative mode).
Will There Be Another Killer Flu? Are We Preparing?
Tumpey 2002 study says yes Viral resistance has been shown Virologists say it is inevitable We won't know until it happens
Virion Structure of ssRNA Phages
Typically comprise 180 molecules of major coat (capsid) protein (CP), one molecule of maturation (A) protein, required for infectivity and maturation, and a linear highly structured ssRNA genome of about 3500-4200 nucleotides
What is virion subunit vaccine?
Usually a subset of viral proteins
1.Is lysogeny a type of non-productive infection?
Yes
3.Can Tamiflu/oseltamivir be used to treat influenza virus infection?
Yes, they inhibit the function of the N protein which cleaves sialic acid on host cells to prevent clumping of viral particles at the host surface --> viral particles clump at host cell surface, reducing viral spread
5.Does HBV genome replication require a primer? If yes, what's the nature and identity of the primer?
Yes, the primer is the protein domain of the P protein
Can Tamiflu/oseltamivir be used to treat influenza virus infection?
Yes. It inhibits neuraminidase activity
Oncogene
a gene that has the potential to convert a normal cell to a cancerous or transformed cell.
More on the EBV episome
acts as a template for viral pregenomic
CRISPR system:
an adaptive immunity against bacteriophages
Carcinogen
any substance, radionuclide or radiation, that is an agent directly involved in causing cancer
Proto-oncogene
cellular genes that promote the normal growth and division of cells
3.Describe the genome constitution of phage lambda λ.
dsDNA genome with cohesive ends, which allows the genome to circularize for enhanced staility
2.Describe how HBV enters host cell.
endocytosis
4.How do influenza viruses enter host cell?
endocytosis
Tumor suppressor genes
genes that suppress or inhibit the conversion of a normal cell into a cancer cell. These genes cause cancer when they are turned off.
8.Describe how HBV genes are expressed? (hint: which host enzyme transcribes viral mRNAs? How many types of mRNAs produced? Do all viral mRNAs have the same 3' ends? How many types of proteins are produced by HBV?)
host enzmye: host RNA polymerase II, 4 types of mRNA produced, all transcripts are capped at 5 end and polyadenlyated at 3 end, so common 3 end
Cancer
in a human or an animal a cancer is a malignant tumor and involves continuous proliferation of a clone of cells derived from one of the body's normal cells.
Oncogenecity (tumorigenesis)
in vivo development of tumors
Influenza virus nomenclature
influenza Type/Species isolated from/ Location of isolation/strain designation/ Year isolation/ HN subtype Example: A/Chicken/Mexico/3/ 94 / H5N2
4.Describe how the P protein of HBV is translated.
leaky scanning
Transmission of HBV Infection
mother to baby Transfusion (blood, blood products) Contaminated needles and syringes Organs and tissue transplantation child to child
Known biological events that can trigger the transformation of normal cell into cancer cell:
mutation, activation of oncogenes and inactivation of tumor suppressors.
7.Does the formation of covalently closed circular DNA (cccDNA) of HBV occurs in cytoplasm?
no it occurs in the nucleus
5.Where do Influenza A viruses replicate their genomic RNAs within host cells (hint: cytoplasm or nucleus)?
nucleus
2.Describe the structure feature and genome constitution of phage jX174, phage T7 Phi6 (ϕ6) and phage MS2.
phage jX174: naked single-stranded DNA virus. The genomic DNA is circular. F protein: form main shell, G protein: forms spikes H protein: on each spike, acts as pilot, J protein: + DNA binding protein T7: large dsDNA virus with head-tail structure. inear genome, icosahedral head. Phi6 (ϕ6): segmented dsRNA genome packaged in a polyhedral inner core with a lipid-containing envelope. The genome comprises three linear segments: RNA L (large), RNA M (medium), and RNA S (small). phage MS2: plus stranded RNA genome, naked virus
Cell cycle control proteins
qActivation of cell cycle progression -cyclins, cyclin dependent protein kinases (Cdks), Cdk inhibitors • qInhibitors of cell cycle progression - tumor suppressors
4.Studies on replication of which class of viruses led to the discovery of rolling circle replication mode?
single‐stranded DNA phages
Cell transformation
the change in the morphological, biochemical, or growth properties of a cell
3.In HBV virion, a P protein is often covalently linked to which strand of viral genomic DNA?
the minus-stranded genomic DNA
LISTEX P100
used in cleaning rooms of cheese, meat, and poultry processing plants to inhibit the growth of Listeria on products such as lunch meat and hot dogs -will not be declared as an ingredient on the label of a treated product
Oncogenic virus
viruses that are able to cause cancer.
Metastasis
when a cell or clump of cells separates from a tumor and spreads to another location.
Effects of Antibody Binding to Virions
§ Release of nucleic acid from virions § Prevention of virion attachment to cell receptors § Note: virus attachment sites of most picornaviruses are in deep canyons, thus are inaccessible to antibodies § Release of virions that have attached to cell receptors § Inhibition of entry into the cell by coating fusion proteins on an enveloped virion § Inhibition of genome uncoating
Why study on bacteriophage is important
§Bacteriophages are a Threat to Fermentation and Pharmaceutical Industries §Bacteria are used in a variety of fermentation processes to produce food, drugs, alcohol etc. §Attacks by bacteriophages can results in considerable economic losses § §Bacteriophages can be used to destroy biofilms and to treat human diseases caused by bacterial pathogens §Biofilms causes environment and health problems §Some bacterial pathogens are resistant to antibiotics § §Bacteriophages can be used as disinfectants in food industry §e.g. LISTEX P100, a cocktail of 6 phages
Hepatitis B Virus (HBV)
§Causes chronic hepatitis which can progress to cirrhosis and liver cancer (HCC or hepatoma). § §Infects the hepatocytes of the liver. § §HCC tumors in patients infected with HBV usually harbor integrated viral DNA. § §HBV vaccine was the first vaccine to prevent a "cancer."
Behaviors of Progeny HBV Nucleocapsids
§During (+) DNA synthesis a nucleocapsid can either migrate to the nucleus to increase the pool of cccDNA or it can undergo a maturation event that enables it to bud through a membrane containing virus envelope proteins § DNA synthesis ceases on budding, as the nucleocapsid is cut off from the pool of nucleotides in the cytoplasm, resulting in incomplete (+) DNA in the virion
Epstein-Barr Virus and Cancers
§First isolated from B cell tumor which is consistently infected with EBV. § §In B tumor cells, chromosome rearrangement results in extremely high level expression of c-Myc, which often causes cancer. § §Chromosome rearrangement can also occur in the absence of EBV. § §EBV is also associated with nasopharyngeal carcinoma which has region specific distribution. § §EBV was often found to exist as covalently closed circular DNA in nucleus.
HBV Genome Organization
§Four ORFs produce seven proteins, very efficient coding strategy. § § The P ORF, which occupies about 80 per cent of the genome, overlaps the C and X ORFs and the entire S ORF is within the P ORF. § § The L proteins are produced in two different conformations that have different functions. § § All the regulatory sequences, such as promoters, are within protein coding sequences. •Translation of the S region -> the S protein; pre-S2-S -> the M protein; the complete ORF -> the L protein. Similar translation mechanism is used for the pre-C-C region translation. • •Host proteins play roles in the replication of HBV, including enzymes, transcription factors and chaperones (e.g.HSP90).
HBV Reverse Transcriptase Activity
§Heat shock proteins associate with RT, allowing it to form into an active conformation § §Active viral RT converts the pregenomic RNA into DNA inside the particles § §Unlike retroviruses, HBV RT occurs by protein-priming as opposed to RNA priming § §Precise replication only occurs inside of intact nucleocapsids
Host Cells for HBV Replication
§Hepatocytes (liver cells) are the host cells for HBV in the body. § §Only primary cell cultures of human hepatocytes support replication. None of the established cell lines derived from liver tumours can be infected by HBV virions ( although some cell lines can be infected using HBV DNA through transfection, not allowing for study of the processes of entry and uncoating). § §Some animal hepadnaviruses, such as the woodchuck and duck HBVs, replicate in cell lines.
HBV Capsid
§Icosahedral symmetry §Has holes in it and short spikes protrude from its surface §Constructed from dimers of the C (core) protein §The C terminus of the C protein is highly basic due to the presence of a large number of arginine residues, which mediates the binding to virus genome.
1997 Avian Flu Scare: H5N1
§May, 1997, 3-year-old boy in Hong Kong died § §Influenza A isolated from the boy but the H subtype could not be identified §Later confirmed to be H5 §H5 not known to infect humans before (subtype isolated from birds only) § §The "jump" of an avian strain directly to humans had never happened before § §Same H5N1 strain was killing chickens
Genetic Variation
§Mutations are common during viral replication § §Viral RNA dependent RNA polymerase lacks proofreading and correction ability § §Two processes, antigenic drift and antigenic shift, mediate genetic variation that causes new strains of influenza to appear
HBV Genome Release and Formation of cccDNA
§Once the virus genome is free in the nucleus it is converted into a circular DNA molecule. The covalently bound P protein is removed from the 5 end of the minus strand, which is shortened to remove the third strand of the triple-stranded region. § §The RNA is removed from the 5 end of the plus strand, while DNA synthesis at the 3 end makes the entire molecule double-stranded. The ends of each strand are then ligated to form covalently closed circular DNA (cccDNA). § §The virus DNA is not replicated in the nucleus, but more copies are brought into the nucleus later in the replication cycle.
Lessons Learned from the 1997 H5N1 Hong Kong Flu
§Only 18 human cases in 1997 §33% fatality rate. Human-to-human transmission is rare §1918 flu was about 4% fatal in the U.S § §It is possible that a single insertion (mutation) could enable the virus to spread much more efficiently to the brain and heart. § §The most important control measures are §rapid culling of infected or exposed bird and proper disposal of carcasses §quarantining and rigorous disinfection of farms
Domains of the HBV P Protein
§Reverse transcriptase activity. § Ribonuclease H (RNase H) activity. § DNA-dependent DNA polymerase activity
Immunological Memory & Viral Vaccine
§Some B cells and T cells can survive as memory cells long after the first or subsequent encounters with viruses. § Memory cells can be reactivated from a resting state if they encounter the same antigen again. These cells are the basis of immunological memory, which can be formed as a result of both natural infection and vaccination. § Therefore, the purpose of most viral vaccines is to induce long-term immunity against the virus by establishing immunological memory that will be triggered if the virus ever invades the body.
Synthesis of HBV DNA by Reverse Transcription
§The pregenome RNA acts as template for DNA synthesis § §The terminal protein domain of P acts as the primer for the initiation of minus-strand DNA synthesis. A covalent bond is formed between the -OH group of a tyrosine residue near the N terminus of P and the first nucleotide § §Initially a 4-nucleotide (−) DNA is synthesized then it is transferred to a complementary sequence in DR1 near the 3 end of the pregenome § §The RNase H activity of the P protein degrades the pregenome RNA from the RNA:DNA duplex § §All the RNA is removed except for a remnant of 15-18 bases including the cap. The -OH group at the 3 end of the RNA remnant acts as the primer for the synthesis of (+) DNA
Cell Entry: Endocytosis
§The virion is endocytosed then the nucleocapsid is released from the endosome by fusion of the virion and endosome membranes. § §During uncoating, viral cores are released into the cytoplasm. § §The nucleocapsid may enter the nucleus through nuclear pores, since HBV capsid has been shown able to do so.
Release of Influenza Virions
§Virions are released by budding § §The viral N protein cleaves the sialic acid on host cells to prevent clumping of viral particles at the host surface § §Zamanivir (sold as Relenza) and oseltamivir phosphate (sold as Tamiflu) inhibit the function of N
Early Cancer Research on Rous Sarcoma Virus (RSV)
•1908 Wilhelm Ellerman and Olaf Bang demonstrated that "filterable agents" could produce tumors in chickens. • •1911 Peyton Rous demonstrated a bacteria-free filtrate caused sarcomas in chickens. -The agent was Rous Sarcoma Virus, a retrovirus.
Bacteriophage Structure
•4 basic shapes or symmetries -Binary (head and tail structure) -Icosahedral (also called cubic) -Helical (filamentous) -Pleomorphic •The majority of bacteriophages contain a head and tail structure
Regulation of Gene Expression in ssRNA Phages
•A complex secondary structure controls gene expression via differential access of the ribosome binding sites (RBSs) to host ribosomes during translation. • •Initially the RBS for the coat protein gene is accessible, whilst those for the maturation (A) and the replicase gene are not. • •The replicase gene is expressed only after the coat protein gene has been translated. • •Translational coupling between coat protein and replicase genes involves the Min Jou (MJ) interaction in which a sequence just upstream of the replicase gene RBS is base-paired to an internal sequence of the coat protein gene. • •Passage of ribosomes beyond the first half of the coat protein gene temporarily melts this interaction, opening up the RBS for translation of the replicase gene.
Phage T4
•A virulent T-even phage with linear dsDNA genome of ~169 kbp. T4 provided evidence of gene splicing through the presence of introns in the genome. • •T4 tail fibres, pins and baseplate are involved in binding to the lipopolysaccharide receptor of the E. coli host. • •The empty capsid remains extracellular after the viral genome is injected into host cell. • •Host RNA polymerase stops recognizing host promoters and instead uses phage promoters for T4 gene expression, and the bacterial genome is degraded and recycled in phage DNA synthesis. • •The genome is AT-rich and contains modified bases in the form of 5-hydroxy-methyl-cytosine, rather than cytosine, which protect the phage DNA from many host restriction systems and from phage-encoded nucleases. • •Has been suggested as possible therapeutic agents in, for example, the treatment of E. coli diarrhoea.
Phage T7(with short, non-contractile tails)
•A virulent T-odd phage, with linear genome, icosahedral head. • •T7 early genes are transcribed by the bacterial RNA polymerase. • •One of these early gene products is T7 RNA polymerase, a very active enzyme that takes over transcription of T7 genes. • •Late genes are transcribed from strong T7 promoters. • •T7 RNA polymerase recognizes only T7 promoters. • •Degradation of the bacterial chromosome during infection provides the nucleotides for T7 replication, which uses T7 DNA polymerase.
mRNA Synthesis and Replication of Virion RNA
•After the viral RNPs enter the nucleus, mRNA synthesis begins • •Cap-snatching process -Viral PB2 protein binds to the cap structure along with a short string of nucleotides of host mRNA in the nucleus of the cell.
Epstein-Barr Virus (EBV)
•Also known as Human Herpes Virus 4 (HHV-4) • •Named after Anthony Epstein and Yvonne Barr, who isolated EBV from lymphoma samples collected by Dennis Burkitt (1964). -The lymphoma was called Burkitt's Lymphoma.
KSHV and Cancer
•Also referred to as Human Herpesvirus-8 (HHV-8) •Causes Kaposi's sarcoma -rare skin cancer. •Virus discovered as the cause of Kaposi's sarcoma in 1994 by Patrick Moore and Yuan Chang. •Kaposi's sarcoma occurs most often -in elderly men of Mediterranean, Middle Eastern or Eastern European -AIDS patients (50% risk).
Ribavirin
•Analogue of guanosine, used for the treatment of infection with several RNA viruses, especially persistent infections with HCV. •Combined treatment with ribavirin and α-interferon has eradicated HCV infection in many patients. •Also being recommended for treatment of young children infected with respiratory syncytial virus. •The drug is delivered to the patient in the form of an aerosol for 12-18 hours daily. •Disadvantage: the mode of delivery is expensive and inconvenient •The mode of action of ribavirin is not completely understood and there may be more than one mode of action as it has been shown to inhibit viral RNA synthesis and mRNA capping.
Good Vaccines Stimulates both Humoral and Cell Mediated Immunity (CMI)
•Antibody is primarily aimed at surface capsid or env proteins of free virus •CMI can be aimed at internal proteins expressed with MHC1 and these may be more conserved among subtypes
Hepatitis B
•Average incubation period is 80 days. •30% of individuals have no signs and symptoms •If symptoms occur, they are similar to hepatitis A + joint pain. •Chronic hepatitis B infections occurs in 5-10% of cases. •Chronic infections lead to: -Cirrhosis of liver -Hepatocellular carcinoma (HCC) or liver cancer (Duck HBV, on the other hand, is non-pathogenic in its natural host)
Overview of Bacteriophage Infection
•Bacteriophages adsorb to the surface receptor molecules of bacteria during the first step of infection -Receptors may be: •Pili •Proteins •Oligosaccharides •Lipopolysaccharides (LPS)
Icosahedral ssDNA phages
•Belong to the family Microviridae (micros='small'). • • •Provided the first evidence for overlapping genes and revealed the economy of genetic coding. • •Studies on replication of these phages led to the discovery of rolling circle replication and to the identity of various genes encoding proteins for host DNA replication
Virion Maturation and Assembly
•Capped mRNAs exported from nucleus are translated by ribosomes in the cytoplasm • •H, N, and M2 are folded and glycosylated and transported to the trans Golgi network and cell inner surface where assembly takes place • •One copy of each genome segment is packaged into the virion.
Characteristics of F-specific Filamentous Phage Genomes
•Circular ssDNA of ~6400 nucleotides. • •The genes are tightly packed on the genome, with a number of intergenic (IG) regions. • •The genome encodes 11 proteins. • •Genes are grouped by function: genes II, V and X for phage DNA synthesis; genes III, VI, VII, VIII and IX for capsid structure and genes I , IV and XI for assembly. • •All the genes are transcribed in the same direction and there is a gradient of transcription with genes near a terminator being expressed more frequently.
Live Recombinant Virus Vaccines
•Examples, vaccinia virus expressing the G protein of Rabies virus.
Soluble Virus Protein (HBeAg)
•Found in the blood of some infected individuals. • •Known as hepatitis B e antigen (HBeAg). • •Similar to the C protein, but has 10 extra amino acid residues at the N terminus and lacks 34 amino acid residues at the C terminus. • •The function of HBeAg, like that of the small spheres and filaments, is currently unknown.
Filamentous Single-stranded DNA Phages
•Found in the family Inoviridae (ina ='fibre, filament'). • •Plus strand phages and 'male-specific', infecting E.coli strains containing the conjugative plasmid F by adsorbing to the tip of the F pilus (encoded by the plasmid). • •Filamentous phages do not inject their DNA into the host cell; rather entire phage particles are ingested. • •Do not lyse infected cells, but progeny are continuously extruded through the cell membrane. • •Form plaque-like zones in lawns of sensitive bacteria, since infected cells grow more slowly than uninfected cells.
Ganciclovir
•Guanosine analogue. •The first phosphorylation in a cell is carried out by a virus enzyme(a protein kinase). •Ganciclovir is more likely than aciclovir to become incorporated into cell DNA, therefore a lower SI than aciclovir and can cause significant side-effects, especially reduced numbers of blood cells because of damage to bone marrow cells. •Used to treat infections with cytomegalovirus, as aciclovir is less effective against this virus than it is against the other herpesviruses. •Example diseases treated with ganciclovir include pneumonia and retinitis in immunocompromised patients.
Co-infection of HBV and HDV
•HDV is a defective virus •Requires the presence of a "helper" HBV to replicate -The surface or outer coat of HDV consists of HBsAgs •HDV associated with chronic hepatitis •HDV uses the same cellular receptor as HBV ---------------------------------------------------- HDV: Membrane proteins are originated from the HBV helper virus Circular, ssRNA(-) genome of 1.68 kb. Replication occurs by rolling circle. Single genome being cleaved/ligated by HDV ribozymes Infection results in the highest mortality rate of all the hepatitis infections (20%).
Phylogenetic Tree of Human Papillomaviruses
•HPV infections most common among sexually active adults and adolescents. •Low-risk types cause warts or papillomas (e.g. genital warts). •High-risk types cause cervical, vulva, vagina, anus and penis cancers (e.g. types 16 and 18).
Virus Vaccines
•Harmless agents, perceived as virus enemies. They are molecules, usually but not necessarily proteins, that elicit an immune response, thereby providing protective immunity against a potential viral pathogen.
Genome Replication
•Host enzymes ligate the ends of the genome • •DNA synthesis is completed, gaps are repaired in both DNA strands -Genome is now a closed circular plasmid-like dsDNA molecule called an episome • •HBV DNA does not integrate into the host chromosome (not integrase activity) •Acts as a template for Viral pregenomic RNA transcripts • •Genomic RNA transcripts transcribed by the host RNA polymerase II
Derepression or Induction
•If the repressor loses function (becomes inactivated), viral DNA is excised from the bacterial chromosome and the excised DNA acts like a lytic virus •Spontaneous derepression or induction happens about 1 in every 10,000 cell divisions • •Temperate bacteriophages can carry host genes from one bacterial cell to another in a process called transduction
Immunity
•Infected individuals develop antibodies against the outer proteins of Influenza virus—neuraminidase (N) and hemagglutinin (H) • •Antibodies against H neutralize the virus • •Antibodies against N do not but they do reduce the release of virus from infected cells •If a person has been infected in the past couple of years by a closely related strain of influenza H subtype their antibodies may intercept and neutralize the virus, protecting the lungs.
Historic Review
•Influenza has been described as early as 400 B.C. • •Major epidemics and pandemics of influenza have occurred throughout history. • •Many virologists think another influenza pandemic that could kill millions of humans is inevitable.
Influenza A Life Cycle
•Influenza virus H binds to sialic acid present on glycoproteins of ciliated cells lining the sinuses and airways. • •Virions enter by endocytosis. • •Inside of the endosome, the virion is exposed to a low pH (from 7 to 5). • •The low pH causes H to undergo a conformational change
Phage Lambda (λ) ( long, flexible tail)
•Isometric head containing a linear dsDNA genome of about 48.5 kbp and central tail tip fiber and side tail fibers. • •The genome has 12-base single-stranded complementary 5 ends (cohesive ends) that mediate circularization of the DNA after infection of the host, which protects viral genome from degradation. The cohesive ends are generated due to the mode of packaging of the DNA from concatemers, which are cut asymmetrically at λ cos sites. • •To subvert bacterial restriction-modification activities upon infection, phage λ encodes the Ral (restriction alleviation) protein, which enhances methylation of the viral DNA, thereby alleviating restriction by type I restriction enzymes. • •As a temperate phage, λ can either establish lysogeny, being maintained as a prophage integrated into the bacterial chromosome between the biotin and galactose operons, or multiply lytically to produce progeny virions.
Synthetic Peptide Vaccines
•Made of synthetic epitopes. •Advantage: Compared with traditional vaccines it would be easier to ensure the absence of contaminants such as viruses and proteins. •Induced reasonable levels of neutralizing and protective antibodies in laboratory animals but failed to do so in farm animals. Requires multiple injections and large doses.
Mass Production of Viruses for Vaccines
•Mainly using animal or human cell culture. •Chicken embryos have been used for Influenza virus. -Reassortments with improved infectivity in chicken embryos were created for preparing vaccines against new influenza virus strains
Double-stranded DNA phages
•Myoviridae, referring to phages with contractile tails. Memebers include T2, T4 and T6. • •Siphoviridae, referring to phages with long, flexible, non-contractile tails. Members include T1, T5 and λ • •Podoviridae, referring to phages with short, non-contractile tails. Members include T3 and T7.
HBV Assembly and Release
•Nucleocapsid cores reach the ER • •At the ER, the nucleocapsids associate with viral surface glycoproteins and bud into the lumen of the ER/and or Golgi • •Empty envelopes containing viral surface proteins of the cell are continually shed, along with mature particles
Classification of Influenza Viruses
•Orthomyxoviridae family •3 Types of Influenza: A, B and C •All 3 can infect and cause similar symptoms in humans •Infection with one type does not confer immunity to another type of influenza
Tumor suppressor p53
•P53 halts progression when DNA damaged -to give cell time to repair or -triggers apoptosis of damaged cell by activating Bcl-2, causing mitochondria to release cytochrome C and activate caspase system •If damaged (mutated) cell moves to S phase then it may replicate
Double-stranded RNA Phages
•Phi6 (ϕ6) was the first member of this family to be isolated and has been extensively studied. Phage ϕ6 infects Pseudomonas syringae, by way of the pilus, which retracts to bring the virion into contact with the cell, and the nucleocapsid enters. • •ϕ6 has segmented dsRNA genome packaged in a polyhedral inner core with a lipid-containing envelope. The genome comprises three linear segments: RNA L (large), RNA M (medium), and RNA S (small). • •Uncoating occurs inside the cell and the polymerase, always carried by the virus, is released. • •Transcription of the double-stranded genome involves a phage RNA dependent RNA polymerase. The plus strand transcripts serve as replication templates and mRNAs.
Exit and Primary Sites of Respiratory Infections
•Primary site of infection—tracheobronchial tree, involving nasopharynx •As virus replicates, cilia are destroyed -Cleaning system in the lungs does not work as well -More mucus stays in the airway, clogging them, causing coughing •Destruction of cilia contribute to secondary bacterial pneumonia infections, sinusitis, otitis
Kaposi's-sarcoma-associated Herpesvirus (KSHV) and Cancer
•Readily detectable in kaposi's Sarcoma and homosexual men. • •Copies of the KSHV genome are present as cccDNA in the nuclei of the tumor cells. • •Also linked to primary effusion lymphomas and multicentric Castleman's disease. Both are tumors derived from B cells.
Inactivated Virus Vaccines
•Require that large amounts being injected and it may be difficult to obtain an inflammatory response required for a vigorous response without overdoing it. •Often made by mass producing the virulent virus and then inactivating the infectivity, usually by treatment with a chemical such as formaldehyde. •Challenges: finding the combination of chemical concentration and reaction time that completely inactivates the virus, but leaves its antigens sufficiently unchanged that they can still stimulate a protective immune response. -204 cases of paralytic polio and 11 deaths caused by a batch of inadequately inactivated poliovirus vaccine. •Successful applications: influenza, hepatitis A and foot and mouth disease viruses
Other Viral Proteins Capable of p53 and/or pRB Function Suppression
•SV40 large T (= tumour) antigen (binds to p53 and Rb). • •KSHV latency-associated nuclear antigen (binds to p53 and Rb). • •HTLV-1 Tax protein (binds to p53). • •HBV X protein (binds to p53).
Polyomaviruses and Cancers
•Similar to papillomaviruses but slightly smaller. Natural hosts are mammals and birds. • •Two strains (JK & BK) that infect human • •Induce tumors in newborn hamsters and capable of cell transform in cell culture. • •Linked to brain cancers. • •SV40 is one of the most extensively studied polyomaviruses
Post-translational Modifications
•Some of the envelope protein molecules become glycosylated and L is myristylated at the N terminus. • •Myristylation of L is not required for efficient virion assembly but virions produced in its absence are not infective. • •Initially all the L protein N termini are on the cytoplasm side of the endoplasmic reticulum membranes, but after translation about 50 per cent of the N termini are moved through the membranes into the lumen of the endoplasmic reticulum. The cell heat shock protein Hsc70 may play a role in this process.
HBV Transcription
•The cccDNA is the template for transcription. • •The HBV genome has four promoters. • •The virus protein X may be a transcription factor, though it appears not to bind to DNA. • •All the transcripts are capped at the 5 end and polyadenylated at the 3 end, using the same polyadenylation signal (TATAAA), therefore a common 3 end. • •Production of the 3.5kb transripts relies on a HBV genomic sequence called positive effector of transcription (PET), which allows the RNA polymerase to continue through the polyadenylation signal on the first pass, but not on the second pass.
Cytokine Storm
•The immune system overreacts toward the pathogen • •Also referred to as systemic inflammatory response syndrome (SIRS), which may explain the devastating nature of the 1918 strain of influenza • •Cytokines signal macrophages to travel to the site of infection, causing damage to the body and organ failure. • •H5N1 Avian Influenza virus also causes SIRS and is at least 50% lethal in humans.
Arrangement of HBV S,M and L Proteins in the Envelope
•The virion envelope contains three protein species, S, M, and L. • •The M and L proteins are longer versions of the S protein, which is the most abundant of the three. • •The surface regions of the envelope proteins constitute an antigen known as hepatitis B surface antigen. •The virus attachment site is near the N terminus of the L protein, but only about 50 per cent of the L molecules have the N terminus on the outside of the virion; the N termini of the remaining L molecules are on the inside bound to the capsid.
Regulation of Gene Expression in ssRNA Phages (cont.)
•Translation of the replicase gene can be repressed by coat protein, ensuring that only a catalytic amount of replicase is translated. The latter half of the infection cycle is then devoted to synthesis of coat protein, which is required in large quantities for virion assembly. •A small amount of lysis protein is synthesized during translation of the coat protein gene, due to an occasional reading frame error. • •Once replicase is synthesized, replication of the genome can occur; the genome thus switches from a template for translation to a template for replication
HBV translation
•Two subsets of 3.5kb RNAs. The shorter subset does not include the start codon for the pre-C sequence and acts as mRNA for the C and P proteins. The ribosome bypasses the upstream start codons by 'leaky scanning' for P protein translation. Thus HBV produces much more capsid protein than polymerase (just as retroviruses control the relative amounts of Gag and Pol proteins synthesized). •The shorter subset of 3.5 kb RNAs can also act as pregenomes. • •HBeAg is translated from the longer subset of the 3.5 kb mRNAs, which include the start codon for the pre-C sequence. After translation HBeAg is secreted from the cell. • •The M protein is translated from the longest subset of 2.1 kb mRNA, whereas the S protein is from several shorter subsets of the 2.1kb mRNAs.
Virus-like Particles as Vaccines
•Typically structures assembled from virus proteins, resembling virions but devoid of any nucleic acid and can therefore be deemed safer than vaccines containing attenuated or inactivated virions. •Example 1: Hepatitis B virus vaccine. Produced in recombinant yeast cells that have the gene for the HBsAg inserted into the genome. •Example 2: The major capsid protein of papillomaviruses can self-assemble into virus-like particles that bear the epitopes required for generating neutralizing antibodies. Human papillomavirus vaccines are produced either in insect cells using a baculovirus vector or in yeast cells. An epitope is a short sequence of amino acids critical for inducing an immune response
Live Attenuated Virus Vaccines
•Usually the most effective vaccine, but can be difficult to balance sufficient attenuation so as not to cause disease in any individual with the necessity for sufficiently vigorous replication to induce immunity •Usually a mutant strain of a virus that has been derived from a wild-type virulent strain. Generally, they are less stable than inactivated vaccines •Produce large amounts of virus antigen in the body as the virus replicates, which induce a wide-ranging immune response, including B cells, CD4 T cells and CD8 T cells. •The take of live virus vaccines can be interfered with by concurrent infection with another virus, which is not a problem with inactivated virus vaccines.
Prevention of Virus-induced Cancers
•Viral transmission prevention (particularly important for viruses transmitted by sex and in blood). • •Vaccination. Success has been seen for HBV. • •Elimination of persistent infection using interferons and antiviral drugs.
mRNA vaccine
•high potency •capacity for rapid development •potential for low-cost manufacture •safe administration