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Lecture 3 Viral Genomes.pptx
- 1. Virus genomes By: Dr.M. Shahzad Iqbal Data given in footnotes of the slides is included in exam
- 2.
- 3. Expression of ViralGenomes Routing of viral genome expression through mRNA. • Route I is transcription of dsDNA usually by the host DNA-dependent RNA polymerase; • route II is transcription of ssDNA to give a dsDNA template for I (e.g., geminiviruses); • route III is transcription of dsRNA, usually by virus-coded RdRp (e.g., reoviruses);
- 4. (AND) Expression ofViral Genomes route IV is replication of (+)-strand RNA via a (−)- strand template by virus-coded RdRp—the viral (+)-strand is often the template for early translation (the (+)-strand RNA viruses); route V is transcription of (−)-strand RNA viral genome by virus-coded RdRp (e.g., Tospoviruses); route VI is reverse transcription of the RNA stage of retro- and pararetroviruses leading to a dsDNA template for mRNA transcription (for pararetroviruses the input viral dsDNA can be the template.
- 5. Overview of Replication and Expression of theViral Genome
- 6.
- 7. 1. DNA viruses– Class I: dsDNA • many host enzymes can be utilized for replication and/or protein production • dsDNA → mRNA → protein, with a DNA-dependent RNA- polymerase • host ribosomes used to make proteins
- 8. The double-stranded DNAviruses Replication of this class, and of all DNA viruses, occurs in the host cell nucleus, although there are some exceptions (e.g., poxviruses). Examples of this class include the Adenoviruses and Herpes viruses. Transcription of viral DNA to produce mRNA occurs in coordinated stages. these correspond to immediate early, early, and late stages of gene expression. Progress through successive stages is dependent on production of viral genome- encoded factors that initiate production of later stage proteins. Replication of viral DNA during the early stage of infection is catalyzed by host polymerase. Expression of late gene products provides structural proteins and enzymes that mediate packaging of replicated genetic material into viable progeny.
- 9. 1. DNA viruses- Class II: ssDNA
- 10. The single-stranded DNAviruses Nuclear replication of this class proceeds from a double-stranded intermediate, or replicative, form of the genomic DNA. The double-stranded replicative form of viral DNA may be produced from the single- stranded template through usurping the DNA repair enzymes of the host cell. In some cases, replication of the double-stranded intermediate occurs through a “rolling-circle” process, resulting in a single linear DNA containing multiple viral genomes. Although genomic DNA is synthesized, viral proteins required for packaging and production of mature particles are produced. In addition to structural proteins, these include a nuclease that cleaves individual genomes during packaging into mature particles.
- 11. Rolling-circle replication The replicativeform can be used for rolling-circle replication, where one strand is nicked and replication enzymes are used to extend the free 3’ end. As a complementary strand is synthesized around the circular DNA, the 5’ end is peeled off, leading to a displaced strand that continues to grow in length.
- 12. 1. DNA viruses- Class VII: DNA viruses that use reverse transcriptase
- 13. DNA viruses -Class VII: DNA viruses that use reverse transcriptase The hepadnaviruses contain a DNA genome that is partially double-stranded, but contains a single-stranded region. After gaining entrance into the cell’s nucleus, host cell enzymes are used to fill in the gap with complementary bases to form a dsDNA closed loop. Gene transcription yields a plus-strand RNA known as the pregenome, as well as the viral enzyme reverse transcriptase, an RNA-dependent DNA-polymerase. The pregenome is used as a template for the reverse transcriptase to produced minus-strand DNA genomes, with a small piece of pregenome used as a primer to produce the double- stranded region of the genomes.
- 14. Double-stranded DNA withRNA intermediate This class is characterized by the hepadnaviruses and is not as well understood as other classes. Release of viral DNA genome into the nucleus of the host cell initiates transcription of mRNA. This viral mRNA serves both for translation of viral proteins and as a template for replicating the viral genome. Replication of the mRNA genomic template is catalyzed after packaging into capsids. These capsids contain viral reverse transcriptase/polymerase capable of copying the mRNA intermediate and producing a double-stranded DNA genome.
- 15. 2. RNA viruses- Class III: dsRNA
- 16. The double-stranded RNAviruses Double-stranded RNA genomes are unique in that they may be poly-cistronic or composed of multiple segments of double- stranded molecules. Transcription of each segment results in production of mRNA encoding a single protein. The protein coat of these viruses contains its own RNA-dependent RNA polymerase, which begins transcription of the viral genome shortly after infection within the cytoplasm of the host cell. Protein-coding mRNA is translated into structural proteins. Packaging of (+) strand RNA from early transcription is followed by secondary transcription of the second RNA strand within the capsid of the maturing viral particle.
- 17. 2. RNA viruses- Class IV: +ssRNA
- 18. Single-stranded sense-strand (+form) RNA viruses In contrast to double-stranded RNA viruses, single-stranded viruses contain a single polycistronic RNA genome. Upon infection, the RNA genome may serve as mRNA and may be translated directly into a single polypeptide that is cleaved to individual proteins by cytoplasmic proteases. Coats of these RNA viruses contain their own RNA-dependent RNA polymerase that synthesizes the complementary (-) RNA strand within the cytoplasm. Late synthesis of structural proteins is associated with increased RNA polymerase production of (+) strand RNA. These polycistronic RNAs are packaged in mature particles prior to release.
- 19. 2. RNA viruses - ClassV: -ssRNA
- 20. Single-stranded anti-sense-strand (- form)RNA viruses Genomes of these viruses may be either mono- or poly-cistronic. In both cases, the (-) strand genomic RNA may be transcribed to mono-cistronic mRNA by a viral RNA polymerase contained within the viral capsid. Cytoplasmic transcription and translation of early regulatory proteins is followed by structural protein production. Through mechanisms that are poorly understood, the late stage of infection is associated with secondary transcription of full-length progeny (-) form RNA. This progeny RNA is packaged into mature phage.
- 21. 2. RNA viruses- Class VI: +ssRNA, retroviruses
- 22. Single-stranded (+) senseRNA with DNA intermediate This class comprises the retroviruses, including the Human Immunodeficiency Virus, or HI V. The (+) strand genome of these viruses does not serve as mRNA, but instead it provides a template for production of DNA by viral reverse transcriptase. This enzyme is multifunctional and catalyzes synthesis of the second viral DNA strand. Retroviral genomes contain special motifs termed “long terminal repeats.” Upon import of viral DNA to the nucleus, long terminal repeat domains pair with host genomic DNA, which favors covalent incorporation of viral DNA into the host genome. This incorporation is catalyzed by a viral “integrase” enzyme. The integrated viral genome is known as a provirus and is replicated with the genomic DNA of the host cell upon cell division. The provirus may also be expressed by the host cell transcription machinery to mRNA encoding viral proteins required for replication. Transcription may also produce to the (+) strand viral genome ready for packaging to mature virus.
Editor's Notes
- #2 Links to source materials https://open.oregonstate.education/generalmicrobiology/chapter/the-viruses/ https://www.sciencedirect.com/topics/immunology-and-microbiology/virus-genome
- #3 Viral genomes consist of DNA or RNA only, never both. DNA and RNA molecules can be double stranded or single stranded, linear or circular (Fig. 1.6), segmented (composed of multiple pieces of nucleic acid) or nonsegmented. The meaning of the term genomic segment could be confusing as its use is driven by both nomenclature principles and historic reasons. Strictly speaking, a genome segment is an individual and unique piece of nucleic acid among multiple pieces comprising one whole viral genome. For example, the influenza A virus has segmented genome comprised of eight ssRNA segments (Fig. 1.5). Herpesviruses, which have nonsegmented genomes composed of one linear dsDNA molecule, have the so-called UL (unique long) and US (unique short) segments, corresponding to regions/portions of their genomes flanked by repeats. The HIV genome can be somewhat confusing too with each virion carrying two copies of the same ssRNA molecule. The HIV genome is considered nonsegmented, and the two ssRNA molecules are called copies not segments. In many viruses the genome ends contain repeated sequences, chemical modifications, or secondary structures, which often have regulatory functions. Genomes are tightly packed inside the capsids and frequently the genome and the capsid are collectively called nucleocapsid. Amazingly, viruses are able to execute productive infection and of course make us sick with very limited genetic information. The flu virus genome, for example, contains only 15,000 nucleotides. For comparison the human genome is 3,200,000,000 nucleotides or approximately 200,000 times longer. Needless to say, viruses have to be superefficient, in their quest to invade the host cell and to propagate. Bacteriophage Qβ is among the smallest RNA viruses with a genome built from 4217 nucleotides and only 4 genes. Among the smallest known animal DNA viruses is TT virus whose genome is comprised of less than 4000 nucleotides and 4 predicted genes. On the opposite side of the scale is the giant Megavirus chilensis with genome as large as 1.3 MB coding for 1000 genes. What functions viral genomes code for is a tantalizing question. Analysis of viral genome sequences revealed that approximately 80% of the viral genomes code for virus-specific genes, many of which have no known homologues or known function.
- #4 Viral genomes are expressed from mRNAs that are either the nucleic acid of positive-sense [(+)-sense] ssRNA viruses or transcripts from negative-sense [(–)-sense] or dsRNA or from ds or ss DNA viruses. Baltimore (1971) pointed out that the expression of all viral genomes, be they RNA or DNA, ss or ds, (+)- or (–)-sense, converge on the mRNA stage. RNA-dependent RNA polymerase
- #5 When the encapsidated virus particle enters a susceptible plant cell, the genome must be released from the relatively stable capsid required for movement from host to host. Once the genome becomes available, it can be translated directly if (+)-sense ss RNA is present, or else the formation of mRNA can commence. As will be described in Section V.A, expression of the viral mRNA faces various constraints imposed by the eukaryotic translation system.
- #6 Viral genomes comprising DNA are most often replicated in the nucleus of the host cell, whereas replication of those comprising RNA typically occurs in the cytoplasm (Fig. 2). In both cases, gene expression to produce viral proteins occurs in the cytoplasm where the protein synthetic machinery of the host cell may be utilized. The basic requirements for replication of viral genomes are the same as those for the infected cells. These include a short nucleic acid primer with a free 3′-OH group that must be available for extension by a nucleic acid polymerase. In many cases, limited packaging space places a premium on genetic efficiency, which is reflected in the evolution of diverse strategies for parasitic use of host enzymes for genome replication and expression of viral proteins. Although viral appropriation of host cell enzymes may reduce the number of metabolic steps that can serve as targets for therapeutic agents, dependence on the activity of unique, viral-encoded enzymes provides such opportunities. For example, viruses like the human immunodeficiency virus (HIV) that have genomes comprise RNA must supply their own polymerase as the cells they infect only produce enzymes capable of copying DNA templates. Targeting of the viral polymerase, as with drugs such as azidothymidine (AZT), enables selective inhibition of this process, minimizing host cell toxicity. Another example is the selective inhibition of herpesvirus-encoded thymidine kinase by acycloguanosine (acyclovir). Mechanisms for replication vary across genome type, with an array of strategies employed. The International Committee on Taxonomy of Viruses currently recognizes six viral taxa, each of which is based on genome type (http://www.uct.ac.za/microbiology/ictv/ICTV.html).
- #7 Since viruses lack ribosomes (and thus rRNA), they cannot be classified within the Three Domain Classification scheme with cellular organisms. Alternatively, Dr. David Baltimore derived a viral classification scheme, one that focuses on the relationship between a viral genome to how it produces its mRNA. The Baltimore Scheme recognizes seven classes of viruses.
- #8 DNA viruses with a dsDNA genome, like bacteriophages T4 and lambda, have a genome exactly the same as the host cell that they are infecting. For this reason, many host enzymes can be utilized for replication and/or protein production. The flow of information follows a conventional pathway: dsDNA → mRNA → protein, with a DNA-dependent RNA-polymerase producing the mRNA and the host ribosome producing the protein. The genome replication, dsDNA → dsDNA, requires a DNA-dependent DNA-polymerase from either the virus or the host cell. The virus often employs strategies for control of gene expression, to insure that particular viral products are made at specific times in the virus replication. In the case of T4, the host RNA polymerase binds to the viral DNA and begins transcribing early genes immediately after the DNA is injected into the cell. One of the early viral proteins modifies the host RNA polymerase so that it will no longer recognize host promoters at all, in addition to moving on to transcribe genes for middle-stage viral proteins. A further modification (catalyzed by middle-stage viral proteins) further modified the RNA polymerase so it will recognize viral genes coding for late-stage proteins. This insures an orderly production of viral proteins. The replication of several dsDNA viruses results in the production of concatemers, where several viral genomes are linked together due to short single-stranded regions with terminal repeats. As the genome is packaged into the capsid a viral endonuclease cuts the concatemer to an appropriate length. There are several animal viruses with dsDNA genomes, such as the pox viruses and the adenoviruses. The herpesviruses have several notable features, such as the link of several members with cancer and the ability of the viruses to remain in a latent form within their host. A productive infection results in an explosive viral population, cell death, and development of disease signs, during which neurons are infected. A latent infection develops in the neurons, allowing the virus to remain undetected in the host. If the viral genome is reactivated, a productive infection results, leading to viral replication and disease signs again.
- #9 Replication of this class, and of all DNA viruses, occurs in the host cell nucleus, although there are some exceptions (e.g., poxviruses). Examples of this class include the Adenoviruses and Herpes viruses. Transcription of viral DNA to produce mRNA occurs in coordinated stages. These correspond to immediate early, early, and late stages of gene expression. Progress through successive stages is dependent on production of viral genome-encoded factors that initiate production of later stage proteins. Replication of viral DNA during the early stage of infection is catalyzed by host polymerase. Expression of late gene products provides structural proteins and enzymes that mediate packaging of replicated genetic material into viable progeny.
- #10 The flow of information for ssDNA viruses, such as the parvoviruses, will still follow the conventional pathway, to a certain extent: DNA → mRNA → protein. But the viral genome can either have the same base sequence as the mRNA (plus-strand DNA) or be complementary to the mRNA (minus-strand DNA). In the former case, a DNA strand that is complementary to the viral genome must be manufactured first, forming a double-stranded replicative form (RF). This can be used to both manufacture viral proteins and as a template for viral genome copies. For the minus-strand DNA viruses, the genome can be used directly to produce mRNA but a complementary copy will still need to be made, to serve as a template for viral genome copies.
- #11 Polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs.
- #16 Double-stranded RNA viruses infect bacteria, fungi, plants, and animals, such as the rotavirus that causes diarrheal illness in humans. But cells do not utilize dsRNA in any of their processes and have systems in place to destroy any dsRNA found in the cell. Thus the viral genome, in its dsRNA form, must be hidden or protected from the cell enzymes. Cells also lack RNA-dependent RNA-polymerases, necessary for replication of the viral genome so the virus must provide this enzyme itself. The viral RNA-dependent RNA polymerase acts as both a transcriptase to transcribe mRNA, as well as a replicase to replicate the RNA genome. For the rotavirus, the viral nucleocapsid remains intact in the cytoplasm with replication events occurring inside, allowing the dsRNA to remain protected. Messenger RNA is transcribed from the minus-strand of the RNA genome and then translated by the host ribosome in the cytoplasm. Viral proteins aggregate to form new nucleocapsids around RNA replicase and plus-strand RNA. The minus-strand RNA is then synthesized by the RNA replicase within the nucleocapsid, once again insuring protection of the dsRNA genome.
- #17 poly-cistronic
- #18 Viruses with plus-strand RNA, such as poliovirus, can use their genome directly as mRNA with translation by the host ribosome occurring as soon as the unsegmented viral genome gains entry into the cell. One of the viral genes expressed yields an RNA-dependent RNA-polymerase (or RNA replicase), which creates minus-strand RNA from the plus-strand genome. The minus-strand RNA can be used as a template for more plus-strand RNA, which can be used as mRNA or as genomes for the newly forming viruses. Translation of the poliovirus genome yields a polyprotein, a large protein with protease activity that cleaves itself into three smaller proteins. Additional cleavage activity eventually yields all the proteins needed for capsid formation, as well as an RNA-dependent RNA-polymerase. The formation of a polyprotein that is cut into several smaller proteins illustrates one possible strategy to an issue faced by many +ssRNA viruses – how to generate multiple proteins from an unsegmented +ssRNA genome? Other possibilities include: subgenomic mRNA – during translation, portions of the viral RNA may be skipped, resulting in different proteins than what is made from the viral RNA in its entirety. ribosomal frame-shifting – the ribosome “reads” the mRNA in groups of three nucleotides or codon, which translate to one amino acid. If the ribosome starts with nucleotide #1, that is one open reading frame (ORF), resulting in one set of amino acids. If the ribosome were to move forward where nucleotide 2 is the starting nucleotide that would be ORF #2, resulting in a completely different set of amino acids. If the ribosome were to move forward again where nucleotide 3 is the starting nucleotide that would be ORF#3, resulting in an entirely different set of amino acids. Some viruses have viral genes that deliberately overlap within different ORFs, leading to the production of different proteins from a single mRNA. readthrough mechanism – a viral genome can have stop codons embedded throughout the sequence. When the ribosome comes to a stop codon it can either stop, ending the amino acid sequence, or it can ignore the stop codon, continuing on to make a longer string of amino acids. For viruses with the readthrough mechanism, they acquire a variety of proteins by having stop codons that are periodically ignored. Sometimes this function is combined with the ribosomal frame-shifting to produce an even greater variety of viral proteins.
- #20 Minus-strand RNA viruses include many members notable for humans, such as influenza virus, rabies virus, and Ebola virus. Since the genome of minus-strand RNA viruses cannot be used directly as mRNA, the virus must carry an RNA-dependent RNA-polymerase within its capsid. Upon entrance into the host cell, the plus-strand RNAs generated by the polymerase are used as mRNA for protein production. When viral genomes are needed the plus-strand RNAs are used as templates to make minus-strand RNA.
- #22 Despite the fact that the retroviral genome is composed of +ssRNA, it is not used as mRNA. Instead, the virus uses its reverse transcriptase to synthesize a piece of ssDNA complementary to the viral genome. The reverse transcriptase also possesses ribonuclease activity, which is used to degrade the RNA strand of the RNA-DNA hybrid. Lastly, the reverse transcriptase is used as a DNA polymerase to make a complementary copy to the ssDNA, yielding a dsDNA molecule. This allows the virus to insert its genome, in a dsDNA form, into the host chromosome, forming a provirus. Unlike a prophage, a provirus can remain latent indefinitely or cause the expression of viral genes, leading to the production of new viruses. Excision of the provirus does not occur for gene expression.