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Introduction to DNA viruses Terje Dokland, Dept

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1 Introduction to DNA viruses Terje Dokland, Dept
Introduction to DNA viruses Terje Dokland, Dept. of Microbiology, UAB BBRB 311, T: Lecture objectives: • Provide an overview of the properties of viruses with DNA genomes, with emphasis on human pathogens • Understand the specific challenges faced by DNA viruses – and advantages of having a DNA genome • Understand the diversity of solutions to these challenges – Replication strategies – Viral life cycles – Relationship between virus and host • Gain insight into how these mechanisms affect viral pathogenesis We will now start talking about DNA viruses more specifically. The purpose of this lecture is to introduce the concept of DNA viruses in general. In particular, I want you to understand the challenges and advantages of having DNA genomes and the range of solutions that DNA viruses have come up with to meet these challenges. The last point is a minor one that I will not really emphasize today.

2 The families of DNA viruses
Group I - dsDNA virus families Order Caudovirales Myoviridae - bacteriophage T4 Podoviridae - bacteriophage P22 Siphoviridae - bacteriophage l Unassigned families: Ascoviridae Adenoviridae Human Adenovirus C Asfarviridae African swine fever virus Baculoviridae Coccolithoviridae Corticoviridae Fuselloviridae Guttaviridae Herpesviridae HSV, Varicella Zoster, Epstein-Barr Iridoviridae Chilo iridescent virus Lipothrixviridae Mimiviridae Mimivirus Nimaviridae Papillomaviridae HPV Phycodnaviridae PBCV-1 Plasmaviridae Polyomaviridae Simian virus 40, JC virus Poxviridae Cowpox (Vaccinia), smallpox Rudiviridae Tectiviridae bacteriophage prd1 Families of DNA viruses that infect humans: Group I - dsDNA virus families Adenoviridae – Human Adenovirus C (respiratory disease) Herpesviridae – Varicella Zoster virus (chickenpox) Papillomaviridae – HPV (warts, cervical cancer) Polyomaviridae – JC virus (PML) Poxviridae – Variola virus (smallpox) Group II - ssDNA families Parvoviridae – Parvovirus B19 (fifth disease) Group III - RNA/DNA families Hepadnaviridae (Hepatitis B virus) Group II - ssDNA families Inoviridae Microviridae bacteriophage fX174 Geminiviridae Circoviridae porcine circovirus Nanoviridae Parvoviridae Parvovirus B19 Group III - RNA/DNA families Caulimoviridae Cauliflower mosaic virus Hepadnaviridae Hepatitis B virus This is a more or less exhaustive list of known DNA virus families as currently recognized by the ICTV (see web site) in all. They are divided here into three groups based on the nature of their genome: dsDNA, ssDNA or a special group of viruses that have DNA genomes but replicate like retroviruses, in particular Hepatitis B virus. (These I will discuss no further today) I have outlined those families that include viruses that infect humans in red. As you can see, there are some very important human pathogens among these viruses, including adenoviruses, herpesviruses, polyomaviruses, poxviruses and parvoviruses. However, most of these families include viruses that do not infect humans; a few of these include viruses that infect other mammals or vertebrates (in bold: ASFV and circoviruses. It would not be a stretch to imagine future human pathogens evolving out of these groups. Some families only infect invertebrates (insects) such as baculo or iridoviruses, but most of these families are bacteriophages, i.e. viruses that infect bacteria. For some reason, there are few families that infect plants. In fact there are higher order, more distant evolutionary relationship between some of these families (e.g. parvo -- micro), that do not appear in this figure. This is a very interesting area, but one that we will not discuss further today.

3 Sizes of DNA viruses • Circovirus • Adenovirus • Mimivirus
genome: ssDNA, 1.7 kb 2 genes capsid diameter: 17 nm • Adenovirus genome: dsDNA, 30-38kbp 30-40 genes capsid diameter: nm • Mimivirus genome: dsDNA, 1.2 Mbp 911 genes capsid diameter: 600 nm DNA viruses come in a range of sizes and level of complexity. At one end of the scale we have circovirus. It is about as simples as it comes, with a 1.7 kb ss DNA genome with only 2 genes (one encoding CP the other a replication protein) and a capsid only 17 nm in diameter. On the other end of the scale is mimivirus, the largest known virus. It has a genome of 1.2 million bps coding for >900 genes and a capsid that is 600 nm in size. You may want to compare this with the smallest cells, Mycoplasma, which is around 500nm in size, has a 580 kbp genome with 470 genes. So here we have a virus that is larger and more complex than many cells. (Of course it is still completely dependent on a host cell for its growth, as are all viruses) Adenovirus represents a more or less average size DNA viruses with … Note, however, that the average size of DNA viruses is far greater than that of RNA viruses. RNA viruses typically have genomes of 5000 nts and the largest, coronavirus, is around 30,000 nt or around the same size as a small adenovirus, and approximately the same size virion as well (100 nm). One of the reasons for this is that DNA replication is less error-prone than RNA replication, thus allowing for larger, stable genomes. As you will see, DNA viruses can have larger and more complex genomes, because they replicate in much the same way as the host cells. • The largest virus, Mimivirus, has a 1.2Mbp dsDNA genome with 911 genes. • Mycoplasma genitialium, a small cell, has a 580kbp genome with 470 genes… • Size of cells: Mycoplasma: <500nm; E. coli: 1-5µm; Eukaryotes: 10–100µm

4 DNA vs. RNA viruses DNA RNA Very stable B-form double helix
dsDNA is rigid Accurate replication large genomes Protected by cell VIRAL DNA IS USUALLY PACKAGED INTO PREFORMED CAPSID SHELLS (PROCAPSIDS) RNA Less stable Mixture of ss and ds forms; extensive secondary structure ssRNA is flexible; dsRNA is rigid Error-prone replication small genomes dsRNA actively degraded by cell RNA MUST BE PROTECTED DURING REPLICATION AND ASSEMBLY! VIRAL RNA USUALLY CO-ASSEMBLES WITH CAPSID PROTEIN There are specific differences between RNA and DNA that has consequences for the structure and life cycle of DNA viruses. DNA, as we all know, forms a relatively rigid double helix, which is chemically very stable. DNA is replicated very accurately by the cellular machinery. As a consequence, DNA viruses can acquire very large genomes. Any DNA is not recognized as foreign by the cell and is in fact protected from degradation by the cell (since it is equivalent to protecting its own genome) RNA is normally single-stranded, but naked ssRNA wil typically form regions of double-stranded structure. Due to the extra OH on the ribose compared to deoxyribose it is less stable. Replication of RNA is more error-prone, carried out by the less accurate viral polymerases (RdRPs). They therefore evolve rapidly, but are limited in the size of the genome they can acquire. More importantly, naked RNA and especially dsRNA is recognized by the cell as foreign and is actively degrated by the cell. Therefore, viral RNA must be protected during viral replication, and is typically sequestered in specialized compartments and protected during assembly. As a consequence, viral RNA usually co-assembles with the capsid protein. In contrast, DNA virus genomes are usually replicated and packaged into preformed capsids or procapsids. This may also be a consequence of the large size and relative stiffness of the genomes.

5 Typical DNA virus life cycle
Typical steps include: Entry Uncoating Nuclear entry Replication Assembly Release Here is an overview of a life cycle for a typical DNA virus. The virus gets into the cell somehow, either by endocytosis or fusion at the plasma membrane or some other mechanism. The genome will have to get to the nucleus, typically involving cellular processes. Uncoating, ore release of the genome from the capsid, may occur in the cytoplasm or after import into the nucleus. Once in the nucleus the cellular machinery may take care of its replication and expression, but usually the virus expresses proteins that are involved in this process. Most DNA viruses are assembled in the nucleus, because that’s where the genome is replciated as well. All PROTEINS are synthesized in the cytoplasm. of course, so the mRNA will have to be exported (using normal cellular processes) and the proteins will be imported back into the nucleus, again using cellular processes and requiring specific tags on the proteins (NLS) to bring them into the nucleus together with other nuclear-resident proteins. Replication inolves cellular (red) and viral factors (green). Release of the virions may involve export back through the nuclear membrane, and posibly budding through the host plasma membrane, or simply lysis of the host cell. Strauss and Strauss (2002) Viruses and human disease

6 DNA virus life cycles DNA is protected by the cell and is transported to the nucleus Many viruses have specific mechanisms for getting the DNA into the nucleus DNA viruses can use replication (DNA > DNA) and transcription (DNA > RNA) machinery of host DNA replication is accurate –> large genomes No need for a viral RNA/DNA polymerase Most replicate and at least partially assemble in nucleus except Poxviruses (+ ASFV, mimi- & iridoviruses) Proteins are synthesized in the cytoplasm and imported into the nucleus Just to emphasize a few important aspects about this process: As I mentioned DNA is protected by the cell and all you need is to get it into the nucleus, which frequently uses the cellular transported machinert. Since cells replicate their DNA genomes and express it usng the traditional DNA-RNA path DNA viruses can use the same machinery. NO NEED FOR RNA-dependent RNA polymerase! More closely related to cells? NOTE: DNA replication is very precise: can make large, stable genomes! Since replication in cells take place in the nucleus, most viruses also replicate in the nucleus. There are some exceptions to this rule, most notable the poxviruses, which replicate in the cytoplasm, and therefore need to create a lot of the replication machinery themselves.

7 Specific challenges for DNA viruses
May require infection of actively growing cells Eukaryotic cells only replicate their DNA in S phase Many cells are frozen in G1 or terminally differentiated If no replication occurs then virus cannot be replicated either Some viruses actively promote cell growth (transformation) Others produce their own proteins for DNA replication Viral latency a prolonged period with no virus production, possibly followed by reactivation virus exists in a plasmid state in the host cell (HSV) integration into the host genome (HPV) Need to enter nucleus (because that’s where the replication and transcription machinery is) except Poxviruses (+ASFV & Iridoviruses) entry of intact virus or uncoating in cytoplasm enter during mitosis Need to exit from nucleus pass through nuclear envelope or lyse the cell So this seems very straightforward. There are however, specific challenges that are faced by DNA viruses too. The problem is that eukaryotic cells are not generally replicating their genomes all the time. Many are frozen in G1 state and do not actively replicate or even express the proteins involved in replication. In this case the virus cannot be replicated either UNLESS it (1) infects actively replicating cells (e.g. blood cells or epithelia); (2) promote cell growth or at least active replication enzymes, or (3) bring in their own replication proteins (DNA polymerase etc). All of these strategies exist in different viruses. Another strategy is to enter into a latent state, in which no virus is produced but the viral genome sits around usually in a plasmid state, but possibly integrated into the host genome for a prolonged period of time (years, maybe), until the host activates replication. e.g. HSV The other problem is that they need to get into the nucleus, and equally important out of the nucleus. Except …

8 Getting access to the cellular DNA replication machinery
The nuclear envelope represents a barrier for the virus to get access to the cellular replication machinery. Solutions: Entry intact e.g. Parvoviruses are small enough (<50nm) to get through the nuclear pore complex (NPC) intact Others are partially unfolded before entry through NPC Disassembly in cytoplasm and transport of genome/protein complex use nuclear localization signals (NLS) Ejection of DNA at nuclear envelope e.g. herpes- and adenoviruses (too large to pass through NPC) Compare to tailed bacteriophages: ejection of DNA through cell wall Some DNA viruses replicate in the cytoplasm Pox-, Asfa- (ASFV), irido- and mimi-viruses very large, complex viruses need to bring all the enzymes required for DNA replication and transcription Cells provide NPCs as a means to transport molecules in and out of the nucleus and viruses utilize this as well. In general this is a poorly understood process. Some small viruses are able to pass through more or less intact. Others disassemble in the cytosplasm and form protein-DNA complexes that are transported into the nucleus by the cellular transport machinery. Still others dock at the nuclear membrane and inject their DNA into the nucleus in a process that resembles the way DNA phages inject their DNA into the bacterial host cell (Fig. on right). A special group of viruses does in fact replicate in the cytoplasm. More about that later. Note that these viruses have to produce all proteins required for replication.

9 DNA replication DNA replication requirements:
A template DNA polymerase A primer (DNA or RNA) Accessory proteins (helicase, RNA nuclease, primase, ss binding protein…) DNA replication is unidirectional From 5’ to 3’ Leading strand vs lagging strand Viral genomes may use RNA primers, DNA hairpins or terminal proteins for priming DNA synthesis What to do at the ends? DNA will get shorter and shorter Eukaryotes use telomerase Prokaryotes have circular genomes (no ends) Viruses have circular genomes or use special terminal proteins Let us first look at replication. What is in general needed for DNA replication? You need a template, a primer, a DNA polymerase and a number of accessory proteins. DNA polymerase (alpha (lagging)/delta (leading) in eukaryotes, DNA pol II in prokaryotes) is unable to start by itself; it needs a primer. The primer can be DNA or RNA. In normal DNA replication the primer is a piece of RNA that is subsequently degraded and then filled in by another?? polymerase (DNA pol I in E coli) The main thing to keep in mind here is that DNA replication is UNIDIRECTIONAL, only from 5’ to 3’. So when you replicate dsDNA at a replication fork, one strand can be replicated as the fork moves (leading strand), the other one is DISCONTINUOUS (lagging strand) and needs to be primed multiple times. Viruses also need primers, but have evolved various strategies, including using RNA primers like the host cell, using a protein attached to the DNA, or self-priming using a terminal hairpin. One problem with replicating a linear genome is that when you are done, there is a last piece of primer at the end. This means that for each round of replication the DNA gets shorter and shorter. Viruses would run out of genome very quickly if this were to happen. Eukarytic cells have telomerase to take care of the ends. Prokaryotes use circular genomes (no ends). They also have either circular genomes, or use special terminal proteins at the ends. 5’ 3’

10 Small, ssDNA viruses: Parvoviridae
5,500 nt linear, self-priming ssDNA, 3 ORFs 18-26 nm naked, T=1 icosahedral virion (60 copies of capsid protein) B19 erythrovirus: causes “fifth disease” (rash, fever); arthritis in adults Several species on animals (cats, dogs, cattle, pigs, minks…) Also adeno-associated virus (AAV) in humans 5’ 3’ Now I will turn to describe some specific examples of DNA viruses. Some of these you will get more detailed lectures on later in this course. The first example is among the simplest DNA viruses, namely the parvoviruses. They are small ≈20 nm T=1 icosahedral viruses consisting of 60 copies of VP1 and genomes of ≈5kb single-stranded DNA. The most important humant pathogen is B19 erythrrovirus, which causes the so-called “fifth disease”. Many animal pathogens - you may be most familiar with FPV which causes feline panleukopenia in cats, and cats get vaccination for this. AAV is a parvovirus that needs co-infection with adenovirus to replicate itself. It does not seem to cause disease in humans by itself, but is important as a potential vector for gene therapy. They integrate into the host genome at specific sites (chromosome 19). B19 cryo-EM reconstruction (Chipman et al. 1996, PNAS 93, )

11 Erythema infectiosum (fifth disease)
characteristic “slapped cheek” appearance • Fifth disease is caused by B19 parvovirus • Mild symptoms in children (rash, fever, clears in 1-2 weeks) – rash is caused by immune response • In adults can lead to polyarthritis • B19 replicates in actively growing erythroid precursor cells (bone marrow) • No vaccine available The main human parvovirus is B19, which causes the so-called fifth disease (after measles, mumps, rubella and chickenpox). Generally mild, but can lead to more severe disease in adults --> due to the robust immune response. Like other parvoviruses, B19 needs to infect actively growing cells, and thus replicates in actively growing erythroid precursor cells in the bone marrow. RASH IS CAUSED BY IMMUNE RESPONSE - not infectious when rash appears. UNLIKE e.g. measles, rubella etc where rash/blisters is caused by replicating virus.

12 Parvovirus life cycle The life cycle of parvovirus is kind of a minimalist system as well, nothing very elaborate. Note the hairpin DNA which is used to prime replication. Assembly, as in most DNA viruses, occurs in the nucleus. The viruses escape by lysing the cell. • Parvoviruses need to infect actively growing cells • Enter nucleus intact (small size) • Exit nucleus/cells by lysis

13 Parvovirus replication
Parvoviruses replicates by a special mechanism that involved the terminal hairpins. The hairpins serve as primers for DNA synthesis One of the viral proteins is involved in this nicking process. Strauss and Strauss (2002) Viruses and human disease

14 Papovaviruses: Polyoma- and Papillomaviridae
Polyomavirus: 45nm capsid “T=7” organization of 72 VP1 pentamers 5,000 bp circular dsDNA genome, 5 genes Large T and small t antigens—transforming proteins JC and BK virus of humans (normally mild) Papillomavirus: 50-55nm capsid “T=7” organization of 72 L1 pentamers Circular, dsDNA genome, 8,000 bp, 9-10 genes Causes warts, cervical cancer The papovaviruses, which used to be considered one family, now consists of two related families of small icosahedral viruses with circular dsDNA genomes. Both viruses have naked, icosahedral capsids (Fig on right is polyomavirus), although the papillomaviruses are somewhat bigger and more complex, with slighly larger genomes. Polyomaviruses do infect humans (JC and BK) but are not generally harmful, except in immunocompromised (HIV, transplant) patients. However HPV is an important human pathogen and is one of the relatively few viruses known to COMMONLY cause cancer in humans. The polyomavirus genome consists of three structural proteins VP1-3, whereof VP1 is the MCP; and two important regulatory proteins large T Ag and small t-ag, which are partially overlapping and are essential for the viral replication. Papillomaviruses are similar -- L1 is the major capsid protein -- but have a few more proteins involved in regulation of transcription and replication and host interaction etc. Many of these genes are overlapping, allowing production of different proteins from the same DNA. Papovaviruses replicate in the nucleus and uses the host DNA polymerase. Virus enter nucleus intact. However, the Large T antigen is needed for DNA replication to occur. This protein has several functions: (1) it binds to the origin of replication to recruit DNA polymerase; (2) it is a transcription factor that activates cells to go into S phase. T antigen is involved in controlling increased transcription from the late promoter (i.e. virus production) and decreased transcription from early promoter (itself). It also interacts with host proteins and changes the properties of the host cell, thus playing a role in cell transformation and tumor formation. Large numbers of capsids accumulate in the nucleus and form inclusion bodies. Virions are released by cell lysis.

15 Polyomavirus replication
Polyomavirus uses host DNA polymerase but needs large T to recruit DNA pol to its replication origin. Large T is also a regulatory protein that promotes/stimulates the transition of cells into S phase. DNA replication is bidirectional (There are two replication forks per circular DNA genome and replication involves leading/lagging strands, Okazaki fragments, DNA ligase, etc.). This process of DNA replication is very similar to that which occurs in the host cell - which is not surprising as the virus is using mainly host machinery except for the involvement of the T antigen.Host histones complex with the newly made DNA. Replication mode also known as “theta” replication Uses host DNA pol but requires “Large T antigen” to recruit it to origin Similar to replication of bacterial genomes bi-directional, RNA primers, leading and lagging strand synthesis Also used by ds/ssDNA bacteriophages Strauss and Strauss (2002) Viruses and human disease

16 Life cycle of polyomavirus
Polyomaviruses are rather cell-specific and the fate of a particular virus is dependent on the interaction between the host cell and the viral T antigen. Polyomavirus can only replicate in the S phase of the cell. So the virus expresses the large T antigen that stimulates expression of enzymes required in replication and forces the cell into S phase. (T antigen is also required for replication of the viral genome, and Tag recruits the DNA pol to replication origin.) In permissive cells, stimulation by Large-T leads to viral production and cell lysis. In some cells types (non-permissive cells) T expression is too low -- fails to recruit DNA pol to origin and virus cannot replicate. Instead the viral genome may integrate into the host genome. This may may switch the virus into the transforming cycle, where constitutive expression of Large T leads to uncontrolled cell proliferation, i.e. a cancerous phenotype. Where a particular cell type is permissive/nonpermissive depends on virus. E.g. polyoma in mouse, SV40 in monkeys. JC only in human fetal glial cells. SV40 • Polyomavirus only replicates in S phase of cells • Large T antigen stimulates entry into S phase (host cell specific) in permissive cells – T antigen also required to recruit DNA polymerase to replication origin • In non-permissive cells, integration of the viral genome may lead to transformation

17 Papillomavirus infection
What I said about polyomavirus is essentially true also for papillomavirus, although the situation is a little more complicated. Most HPV infections are localized skin infections (warts) that clear themselves. Virus may enter through breaks in the skin into a variety of cell types, but moves as naked DNA into the granular layer of the epithellia, where viral growth occurs. The virus then stimulates growth in the granular layer of the epithelia through expression of its E6 and E7 proteins, which then gives rise to the growths known as warts. These are normally benign and self-limiting altough they can persist for a long time. The problem occurs when the virus integrates into the genome in the appropriate cell type, which is what happens with certain high-risk types of HPV in the cervix. Which is why a vaccine (Gardasil) has now been developed against these high-risk types. The virus is tissue-specific (e.g. skin, wart-causing type may not infect mucosa etc.) NOTE: Because the virus is non-enveloped it is HARDY and can persist on surfaces. E6 and E7 bind and inactivate the p53and p105 (Rb) tumor suppressor genes (more of that in Dr Engler lecture). • HPV infects epithelial tissue (skin or mucosal) • Infection may cause warts (stimulation of cell growth in granular layer) • HPV may cauce cervical carcinoma by integrating into the host genome, expression of E6 and E7 oncogenes – inactivate tumor suppressor genes

18 Adenoviruses TP Naked (non-enveloped) capsid, 90nm diameter
30-38 kbp linear dsDNA genome, inverted terminal repeats, genes A 55kDa 5’ terminal protein (TP) acts as initiator for DNA synthesis Adenovirus encodes its own DNA-dependent DNA polymerase 5’ 3’ TP Adenoviruses are among the largest non-enveloped viruses. It is about 90nm in diameter, icosahedral, as shown in the micrographs and the 3D structure below. It has fibres that are involved in infection process. The adenoviruses genome is a linear dsDNA with a protein covalently attached at the 5’ ends for reasons that will become clear shortly. Adenovirus also encodes its own DNA polymerase (even though it replicates in the nucleus)

19 Adenovirus disease Adenoviruses are very common
5-10% of respiratory disease in children worldwide and non-seasonal acute respiratory disease (ARD) (Ad 4, 7) in military rectruits conjunctivitis “shipyard eye” (Ad 8) gastroenteritis (Ad 11, 12) Symptoms: high fever, sore throat, aches, conjunctivitis Species specific human adenoviruses only infect humans Transmission from person-to-person Respiratory, fecal-oral, close contact virus is resistant to inactivation by acid, dehydration and detergents Site of infection: epithelia of respiratory tract, intestinal tract, urinary tract, conjunctiva Virus may spread to and persist for a long time in lymphoid tissues No vaccine is currently in use Vaccination of military recruits discontinued in 1996 Adenovirus are very common and cause a wide range of diseases, including flu or cold-like respiratory symptoms. It is considered

20 Adenoviral conjunctivitis
Adenovirus infection is associated with a large number of diseases, from common cold (acute respiratory disease), pharyngitis, gastroenteritis and diarrhea, and conjunctivitis, which may be of special interest to some of you. Irritation of eye is a risk factor -- I got it after bike riding at night without protective glasses in Singapore with a lot of dust, exhaust and the like.

21 Adenovirus nuclear entry
? Entry into the cell is a complex stepwise entry/disassembly pathway. It is assumed to enter via endocytosis, but it is not clear how it crosses the cell membrane. Adenovirus gets access to the nucleus in a complex and I think not really well understood entry/disassembly process. It is not clear how it passes through the cell membrane. Only a DNA/protein complex enters the nucleus. Only DNA/protein complex enters nucleus (Exits from the nucleus by cell lysis)

22 Adenoviruses use a 5’ terminal protein to prime DNA replication
• There is no lagging strand synthesis in adenovirus, and no DNA/RNA primers are involved • A ss DNA binding protein is required to protect the single displaced strand. • There is no lagging strand synthesis in adenovirus, and no DNA/RNA primers are involved

23 Herpesviruses Large dsDNA viruses
120–230 kbp circular dsDNA At least >70 ORFs, no splicing Enveloped virions nm in diameter: icosahedral nucleocapsid core amorphous tegument layer envelope with glycoproteins Numerous human pathogens Herpesviruses is a very imporant group of viruses medically speaking and includes several human pathogens, like chicken pox, epstein-barr virus etc. There will be another lecture specifically on herpesviruses so I will just mention a few things.

24 Herpesvirus disease Virus Disease Primary target cells Site of latency
Means of spread Alphaherpesviruses Herpes simplex 1 (HSV-1) cold sores mucoepithelial cells neurons close contact Herpes simplex 2 (HSV-2) genital ulcers close contact (STD) Varicella-zoster virus (VZV) chickenpox, shingles respiratory and close contact Betaherpesviruses Cytomegalovirus (HCMV) mononucleosis, birth defects monocytes, lymphocytes, epithelia monocytes, lymphocytes close contact, transfusions, congenital Gammaherpesviruses Epstein-Barr virus (EBV) mononucleosis (glandular fever), lymphoma B cells saliva Kaposi’s sarcoma-related virus (KSV) tumors lymphocytes Here is an overview of some of the most common herpesvirus that infect humans. As you can see they cause a range of diseases, from relatively harmless to serious, some that are very common, others that are rare. Such as chickenpox for example, a common childhood disease, now usually vaccinated against, cause by VZV. Herpes simplex, a common cause of cold sores. Cytomegalovirus, is one of the most common causes of birth defects. I will not go through this in any detail, but one thing that you would want to observe is that these viruses may enter a latent state in sites different from their sites of initial infection, where they can persist for years without producing virus. I will come back to this in a moment.

25 Herpesvirus life cycle
Stage 1 (immediate early): • Penetration and release of DNA in nucleus • Expression of Immediate Early proteins (transcription factors) Stage 2 (early): • Expression of DNA polymerase and other enzymes required for DNA replication • Construction of nuclear factory • Genome replication Stage 3 (late): • Synthesis of structural proteins • Assembly of capsid (in nucleus) • DNA is packaged into preformed procapsids, similar to the process in bacteriophages • Construction of cytoplasmic factory (“Assembly compartment”) • Budding and release of mature nucleocapsids through the nuclear envelope • Tegumentation occurs in nucleus and cytoplasm • Tegumented capsid buds into membraneous compartments • Final assembly and release by exocytosis The herpesvirus life cycle is divided into three stages. The first stage starts immediately after the virus enters the cell. First the genome has to make it into the nucleus. The nucleocapsid is released into the cytoplasm when the viral envelope fuses with the cell membrane. Then the nucleocapsid docks with the nuclear envelope and injects its DNA into the nucleus, much as a bacteriophage injects its DNA into the host cell (bacterium). Once in the nucleus, expression of the immediate early transcripts starts. These genes code for proteins involved in further transcription, i.e. txn factors. These initiate stage two, the early transcription. These are proteins involved in DNA replication, DNA polymerase etc and those involved in the nuclear stage of the assembly process. Initiation of DNA replication marks the start of stage 3, late transcription. Late transcripts include all the structural proteins that are to be incoroporated into the virion. During this stage the DNA is replicated, nucleocapsids are assembled in the nucleus, transported to the cytoplasm by some mechanism which is not quite clear, and assembled with tegument proteins that surround the NC, buds into ER-derived membranes, acquires glycoproteins etc. and finally is release either by exocytosis or by cell lysis. Sometimes viruses are also transferred directly to neighboring cells (to avoid immune detection) and sometimes causing cell fusion (cytomegalovirus).

26 Assembly and DNA packaging in herpesviruses resemble tailed dsDNA bacterophages
DNA replication via rolling-circle mechanism leads to the formation of DNA concatemers Formation of procapsid precursor, using a scaffolding protein DNA is packaged into procapsid through a portal Concatemeric DNA substrate is packaged by a terminase complex Herpesviruses are now considered closely related to the most familiar bacteriophages, the tailed dsDNA bacteriophages such as T4, lambda etc. Strauss and Strauss (2002) Viruses and human disease

27 Viral latency Latency is a hallmark of herpesvirus infections
The viral genome exists as an episome (naked, circular DNA) in the host cell nucleus No virus is produced until reactivation Not the same as persistent infection (continuous viral production) E.g. VZV, which causes chickenpox in children, causes shingles when reactivated in the adult Herpesviruses also undergo an interesting an important process of latency. You have a productive viral infection in peripheral cells. This is cleared by the immune system. For example chickenpox is children, which leads to a rash (epithelial cell lysis). The virus then moves up the neuron and into the nucleus where it stays as an episome, circular DNA for months or years. No virus is produced (which would kill the cell). Until reactivation, virus is produced and released causing renewed viral production. E.g. VZV … Activation may occur when the individual is immunocompromised or for some reason is unable to control the infection. Herpesviruses are not the ONLY ones that undergo latency. Also adenoviruses, polyomaviruses. Knipe and Cliffe 2008

28 Cytoplasmic DNA viruses: the exception to the rule
Some families of DNA viruses replicate in the cytoplasm: Poxviridae – smallpox, vaccinia (cowpox) … Asfaviridae – African Swine fever virus (ASFV) Iridoviridae – insects and lower vertebrates Phycodnaviridae – Paramecium bursaria Chlorella virus (PBCV), infects Chlorella unicellular algae Mimivirus – amoeba These viruses need to synthesize all the enzymes required for DNA replication and transcription consequently, they are large (180–300kbp) and complex (>200 proteins) Replication and assembly takes place in “viral factories” in the cytoplasm So far I have told you that DNA viruses replicate in the nucleus. There is however a group of viruses that replicate in the cytoplasm - the exception to the rule. This group encompasses several families that are probably distantly related, namely poxviruses-the only one with human pathogens, ASFV-just one member that infects pigs, iridoviruses-infect insects and other invertebrates as well as lower vertebrates (frogs, lizards, fish…) but not mammals; a virus that infects algae, and one virus that infects amoeba, which is the largest virus known. These viruses … therefore, they are large and complex In these viruses, replication and assembly both take place in a special region of the cytoplasm called a viral factory.

29 The Poxviruses Many members, infecting vertebrates and invertebrates, divided in several genera: Orthopoxvirus (Variola, Vaccinia, monkeypox) Parapoxvirus (orf; sheep and goat poxvirus) Avipoxvirus (bird viruses) Molluscipoxvirus (Molluscum contagiosum) (NB: chickenpox is not a poxvirus!) Virion: Large (360nm long axis), brick-shaped, multi-enveloped Genome: kbp dsDNA, terminally redundant, inverted repeats DNA replication is self-primed (hairpin) and leads to the formation of DNA concatemers 360 nm The most important from the point of view of human health is of course the poxviruses. Poxviruses are divided into several genera, of which the most important for us and historically are the orthopoxviruses, which include Variola, or smallpox; Vaccinia or cowpox, which gave rise to the first known vaccine -- hence the name, from vacca=cow, and monkeypox. Just remember this: chickenpox is not a poxvirus -- it’s a herpesvirus. These are large and complex viruses; in size among the largest known. Here is a picture of vaccinia virus, 360 nm long, characteristic “brick” shape. Complex structurs with multiple layers of envelopes. The genome is also large, codes for at least a few hundred proteins. Genome is circular and partially double stranded as shown here. Replication is self-primed, similar to parvovirus.

30 Smallpox (Variola)

31 Orf (sheep and goat pox)
localized lesions caused by other pox viruses - virus infects but cannot spread and lesions disappear after a few (3-5) weeks. Caught by direct contact with infected animals (vets, farmers…). Exception is monkeypox, which does spread through the body and leads to smallpox-like disease. Most spread by contact, but smallpox is spread through respiratory route.

32 Replication cycle of vaccinia virus
One of the most studied member of the poxvirus family is the vaccinia virus, as it is safe to work with, once you've survived vaccination. In addition to the genome, the virion contains many enzymes necessary for early stage gene transcription. After entry into the cell, the enzymes packaged in the virion transcribe the early genes. These genes express host modulatory proteins, particularly those for evading immune defenses, many of which are targeted to the cell surface, or secreted. Early genes also express the replication machinery and the intermediate transcription factor. After genomic replication has started, the intermediate genes are transcribed. Finally, late genes are transcribed, which express the structural proteins and the early transcription factors that must be packaged into the virion. This cartoon shows only one path for virion exit. The virus is more interesting that than. We'll come back to virions in a minute. (Moss, B. "Poxviridae: the viruses and their replication" Fields Virology. Eds. D.M. Knipe and P.M. Howley. Philadelphia: Lippincott Williams & Wilkins. pp. 2849– ) 32

33 IMV=intracellular mature virions
Figure 2 Structural changes in viral factories of VV-infected cells membrane-enclosed replication complex (early phase) Viral Factory C = “crescents” IV = immature virus IMV=intracellular mature virions EEV=extracellular enveloped virions This is what it looks like in an actual cell. Here are sections through a cell assembling vaccinia. First you see the formation of this replication center, surrounded by mitochondria. When assembly starts, this replication center changes into a viral factory, which is really a region of the cytoplasm here next to the nucleus. Within this factory, immature particles start to form. These get wrapped in ER and move towards the cell surface eventually forming mature intracellular particles (IMV) and extracellular particles (EEVs). Biology of the Cell Biol. Cell (2006) 97,

34 DNA viruses: Things to consider
What properties of DNA vs. RNA impact the replication strategy of the virus? What challenges does the virus face and what strategies does it employ to resolve these challenges? How does it get into the cell? How does it get into the nucleus? intact or disassembled? How does it replicate its DNA? linear vs. circular DNA primers? What does the virus need to replicate itself? What cellular functions can and/or does it use? dsDNA viruses can take advantage of the cellular DNA replication and transcription machinery Where does it find those functions? most dsDNA viruses replicate in the nucleus What functions does it supply? some dsDNA viruses supply DNA polymerases and enzymes involved in DNA synthesis – why? Cells only replicate their DNA during S phase. Many cells are halted in G1. How does the virus deal with this? infect actively growing cells (parvo) activate the cells (polyoma) viral latency (herpes) co-infect with helper virus (AAV) Take home message: Think about what the virus NEEDS to replicate itself, whether it is a DNA/RNA, ss/ds, enveloped/naked virus. What are the specific challenges it faces? What strategies it uses to get what it needs and face those challenges. What cellular function can or does it use, and what functions does it provide? On a slightly different note, look at differences and similarities between viruses and how those similarities/differences link them together or separate them into specific groups. These are the things I want you to take away from this lecture; more details on specific dsDNA viruses and the diseases that they cause will come in subsequent lectures. You might think about how the virus’ solutions to these challenges may lead to its pathogenicity and how the cell fights that. For example, the fact that viruses may need to activate replication in normally dormant cells may be a path to cancer.

35 Literature and resources
Murray et al Medical Microbiology, 5th ed. (Elsevier Mosby) Chapters 6 and Strauss J.H. and Strauss E.G Viruses and human disease. Academic Press. Shors, T Understanding viruses. Jones and Bartlett. Voet and Voet. Biochemistry. Chapter 31: DNA replication.

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