BIOL 4190 Exam 3

6.Describe how each of the three classes of retrovirus proteins (env, gag and gag-pol) is produced.

((Please see lecture slides for details. Please pay attention to the mechanisms used to ensure that two proteins are produced from a single viral mRNA.)) Gag and pol genes are translated from genome-length mRNAs into gag and gag-pol polyproteins.Most ribosomes terminate translation after synthesis of gag, but some continue the translation making gag-pol--ribosomal frameshift mech to ensure proportions of gag pol---there is a -1 shift in the reading frame from before the gagpol junction (happens 5% of the time) and results in gagpol ENV proteins genes are translated from spliced mRNA in the rough endoplasmic reticulum. The Env protein molecules are then transported to the Golgi complex, where they are cleaved by a host protease into SU and TM molecules.

HIV is a Complex Retroviruses

*need to add picture

Influenza Virus Has Eight RNA Segments

- Polymorphic Virions are 80-100 nm in Diameter. - Filamentous forms may occur. - Prominent 16 nm hexagonal surface spikes - Eight nucleoprotein segments are present inside the envelope Lipid in membranes is of host origin. Three membrane associated proteins. Four core associated proteins. Two proteins are nonstructural.

Mononegavirale

---Negative strand RNA viruses with nonsegmented genome §Minus sense RNA genome is complementary to mRNA. §Genomic RNA is not translatable. §Viral RNA is transcribed into (+)sense mRNA. §RNA alone is not infectious. §Virions contain RNA dependent RNA polymerase. §All negative strand viruses are enveloped. §Members of this order are responsible for some of our most serious diseases.

Cell-free Synthesis of Infectious Recombinant virus

--Picornaviruses are naked viruses that do not need host membrane for virion assembly --Bypass the cell entry step, making it possible to use non-permissive cell for virus reproduction

Describe the major functions conferred by rhabdovirus M protein.

-Forms a layer between the nucleocapsid and envelop -Inhibits transcription of all 3 host RNA polymerase (transcriptase)and blocks intracellular transport of cell RNAs and proteins -Condenses the nucleocapsid into tight coiled helix and links it to a region of the plasma membrane that has copies of G proteins inserted -Has a late domain that binds cell proteins involved in the budding process

Epidemiology

1. Sexual transmission - male homosexuals and constitute the largest risk group in N. America and Western Europe. In developing countries, heterosexual spread constitute the most important means of transmission. 2. Blood/blood products - drug abusers represent the second largest AIDS patient groups in the US and Europe. Haemophiliacs were one of the first risk groups to be identified 3. Vertical transmission - the transmission rate from mother to the newborn varies from around 15% in Western Europe to up to 50% in Africa. Vertical transmission may occur transplacentally route, perinatally during the birth process, or postnatally through breast milk. 4. In 2003, an estimated 4.8 million people (range: 4.2-6.3 million) became newly infected with HIV. This is more than in any one year before. Today, some 37.8 million people (range: 34.6-42.3 million) are living with HIV, which killed 2.9 million (range: 2.6-3.3 million) in 2003, and over 20 million since the first cases of AIDS were identified in 1981.

substrate + ATP +kinase--> ?

ADP + substrate(+P)

Steps in the Assembly of Poliovirus

After Translation and release by the 2A protease, P1 folds and is cleaved by the 3CD protease to form the 5S protomers. The provirion is converted to an infectous form by internal cleavage of VP0 molecules to VP4 plus VP2. *assembly steps are not reversible

ssRNA Phages

All known ssRNA phages are grouped into the family Leviviridae Viruses in this family, such as phage MS2, have genomes with characteristics of bacterial mRNA: the genomes are polycistronic, with all of the ORFs translated by internal initiation

Other dsRNA viruses

All, with exception, are naked virions with icosahedral symmetry The viral dsRNAs are always enclosed within virus protein structures in order not to triggered host antiviral mechanisms

Picornavirus Attachment

Approx. 10% of human rhinoviruses have Low-density lipoprotein as receptors The poliovirus receptor is the glycoprotein CD155. Its function for the host is not known, but it is expressed on many cell types. Only found in humans and some other primate species. Transgenic mice expressing CD155 become susceptible to poliovirus infection

Poliovirus Post-translational Modifications

Because cleavage efficiency varies for different cleavage sites. The amounts of functional proteins produced vary a lot. Example, more capsid proteins, less RNA polymerase

3.Why a 5' end cap structure is not required for picornavirus genomic RNA to serve as mRNA for protein translation?

Because of the internal ribosomal entry site at the 5' end of viral genome

1.Retrovirus genomic RNAs are plus-stranded RNAs with cap at the 5' end and polyA at the 3' end but do not function as mRNA, why?

Because the genomic RNAs are not accessible to protein synthesis machinery after they enter host cell and will soon be converted into double‐stranded DNA

Retroviruses as Gene Vectors

Because they can introduce genes into a variety of cell types, where the genes are expressed at high levels after integration into the cell genomes, some genetically modified retroviruses are used as gene vectors. Usage: expression of genes in cell cultures and for clinical treatments of genetic disorders and cancers. Advantage: they can deliver genes into non-dividing cells and tissues. Caveat: the successes have been tempered by the development of cancer in a few treated patients.

Rotavirus Attachment to Host Cell

Binding of a virion to a cell is initially via sites on the spike proteins (VP4, VP8*, VP5*) and then via sites on the capsid surface (the glycoprotein VP7) Cleavage of the spike protein VP4 to its products VP8* and VP5* by proteolytic enzymes such as trypsin results in much more rapid entry into the cell It is likely that these proteins interact with a number of cell surface proteins; there is evidence that VP5* and VP7 bind to integrins

Binding of Picornavirus to Receptors

Binding of a virion to receptors results in major changes to the capsid structure, namely, the N termini of VP1 move from the interior to the exterior surface of the capsid and VP4 is lost from the virion Binding of free CD155 to poliovirus particles also causes loss of VP4

What's the function of HIV vif protein?

Binds APOBEC3 proteins and brings them to proteasome for degradation

3.What's the function of the primer binding site in retrovirus genome?

Binds host tRNA, which will be used as primer for viral genome replication

Mammalian Diseases Caused by Orbiviruses

Bluetongue disease: very high mortality in domestic & wild ruminants and in wild carnivores. transmitted by a sandfly in which the virus multiplies. Indigenous to Africa but sometimes seen in Europe & in USA. Deer in Southeastern USA may have occasional get Epizootic Hemorrhagic Disease caused by an Orbivirus. African Horse Sickness: Highly lethal disease of dogs, goats, sheep & cattle, elephants, and camels.

1.What's the cell receptor for poliovirus?

CD155

What's the cell receptor for HIV?

CD4

Why HIV Is Resistant to Host Immune Systems ?

CD4 T cells play pivotal roles as helpers for several cell types including B cells, cytotoxic T cell precursors, natural killer cells and macrophages, hence immune responses are impaired. The virus evolves as the infection proceeds, producing new antigenic variants that may not be recognized by the antibodies and T cells present. In latently infected cells the virus is shielded from the immune system.

HIV Receptor & Co-receptor

CD4 can be found on helper T cells and some macrophages, but CD4 T cells are the main target cell of HIV. HIV-1 virion must also attach to a co-receptor, the chemokine receptors, which bind chemokines and mediate leukocyte (white blood cell) trafficking and T cell differentiation. CCR5 and CXCR4 type chemokine receptors on T cells act as coreceptors for HIV-1. C=cysteine, X= any amino acid

Picornavirus Replication

Class IV viruses in Baltimore classification The virus genome can function as mRNA The first virus molecules to be synthesized in an infected cell are proteins

Attachment and Entry of rhabdovirus

Clathrin-mediated endocytosis after a rhabdovirus virion attaches to receptors at the cell surface The G protein spikes are involved both in the attachment to cell receptors and in the membrane fusion The nucleocapsid is released into the cytoplasm after the membranes of the virion and the endosome have fused

Colorado Tick Fever & Coltiviruses

Colorado Tick Fever: Tick-transmitted disease of campers & hikers in Western mountain regions of the USA. Coltivirus Genus: Smooth spherical Capsids with twelve dsRNAs. Vector: Wood Ticks - Virus multiplies in ticks. Adults usually transmit to humans & Nymphs transmit to small rodents. Animal Reservoir: Golden-mantled ground squirrel Disease Symptoms: Acute Onset of Fever and Chills, Severe muscle and headache. Complete but slow recovery normal. Can be more severe in young children. --Infectious virus can be recovered in red blood cells up to four months after infection of adults.

4.What's viroplasm? Are there viroplasms formed during reovirus infection?

Dense discrete regions formed during reovirus infection. They are areas in cytoplasm where virus proteins accumulate. Yes, in rotavirus infections.

Reverse Genetics for Minus-stranded RNA Viruses

Difficulties: Minus-strand RNA is not infectious, so techniques had to be devised that will not only generate virus genomes from cDNA, but also supply the RNA polymerase and the nucleoprotein that coats the newly synthesized RNA Application: Introduction of a defined mutation to investigate the function of a viral gene in the replication cycle and its role, if any, in virus virulence Also being explored as a tool to engineer virus strains with reduced virulence for use in vaccines

Orbivirus Characteristics

Donut-Shaped Capsids with 10 dsRNAs. Virus is transmitted by Ticks & Sandflies and replicates in vectors. Causes Several serious animal diseases.

10.What are the terminal structure feature (cap and polyA tail) of rotavirus produced early and late transcripts.

Early transcripts, but not late transcripts, have cap at the 5' end. Neither early transcripts nor late transcripts have polyA tail

Rotavirus Targets _________ for Replication

Enterocytes (intestine)

Pathogenesis of Rotavirus Infection

Enterocytes on the villi are destroyed as a result of rotavirus infection and this leads to reduced absorption of water, salts and sugars from the gut There is evidence that the tight junctions between cells are damaged by the non-structural protein NSP4, allowing leakage of fluid into the gut. These effects of virus infection, together with the secretion of water and solutes by secretory cells, result in diarrhoea and this can lead to dehydration. There is evidence that on occasion the virus may cross the gut and infect other tissues

Post-translational Modification

Env becomes heavily glycosylated, acquiring an apparent molecular weight of 160 kD. Trimers of Env are formed before the molecules are cleaved to form the envelope proteins gp120 and gp41; the cleavage is carried out by furin, a host protease located in the Golgi complex. Vpu is a membrane-associated protein and is required for efficient budding of virions from the plasma membrane. Some of the virus proteins are phosphorylated, including Vpu and the MA region of some Gag molecules.

Retrovirus Structure

Enveloped 80-110 nm diameter Genome: single-stranded RNA plus polarity 9-10 kb Contains reverse transcriptase

Rhabdovirus Virion structure

Enveloped Helical nucleocapsid Genome: single-stranded RNA minus polarity 11-15 kb

HIV Structure

Enveloped RNA virus, 120nm in diameter Genome consists of 9200 nucleotides (HIV-1) gag core proteins: p15 (vpr), p17 (MA) and p24 (CA), p7 (NC) pol proteins: p16 (protease), p31 (integrase),etc Env proteins: gp160 (gp120:outer membrane part, gp41: transmembrane part)

Describe the structure feature and genome constitution of rhabdoviruses.

Enveloped viruses with minus‐stranded RNA genome. Matrix protein in between envelope and nucleocapsid. The nucleocapsid has helical symmetry. Always carry RdRP, which is used for genome replication and transcription. The genomes have no cap or poly A tail

Rhabdovirus Virion

Enveloped, rod- or bullet-shaped structure containing five protein species: L,P,N, M,G proteins Nucleocapsid of helical symmetry, made of nucleoprotein (N) and viral genomic RNA: one monomer of N protein coats nine nucleotides P (phosphoprotein) and L (large) proteins are associated with N proteins. The L protein is a multifunctional protein that takes up about half of the genome coding capacity. The M (matrix) protein forms a layer between the nucleocapsid and the envelope, and trimers of G (glycoprotein) form spikes that protrude from the envelope

Picornavirus Reverse Genetics

Example: Replacing the glycine residue with alanine at the N terminus of VP0 leads to the inhibition of virion assembly, suggesting that VP0 myristylation is essential for virion assembly to proceed

Transcription Strategy of Viruses with Ambisense Genomes

Examples: Arenaviridae: Both segments of the viral genome are ambisense genomes Bunyaviridae: tomato spotted wilt virus, two of the three genome segments are ambisense

Rabies Virus host and mechanism

Exceptionally wide host range. Infects many mammalian species in the wild. Also infects cell cultures from mammals, birds, reptiles and insects under laboratory condition Infection with rabies virus normally occurs as a result of virus in saliva gaining access to neurons through damaged skin. The infection spreads to other neurons in the central nervous system, then to cells in the salivary glands, where infectious virus is shed into the saliva By estimate rabies causes about 60 000 deaths annually. Most rabies infections of humans are acquired via bites from rabid dogs

Coxsackie viruses

First isolated in 1948 in the US town of Coxsackie (Greene County, New York) Cause a range of medical conditions, including myocarditis (heart disease), meningitis (severe headache and stiff neck) and rashes

HIV is resistant to host immune systems, why?

First. HIV targets and destroys CD4 T cells, which play pivotal roles as helpers for several cell types including B cells, cytotoxic T cell precursors, natural killer cells and macrophages, hence immune responses are impaired. Second, the virus evolves as the infection proceeds, producing new antigenic variants that may not be recognized by the antibodies and T cells present. The third, in latently infected cells the virus is shielded from the immune system.

Influenza Virus: Biology, Ecology & Pathology

Flu was thought to be the "last great plague" on earth. Hippocrates recorded influenza in 400 BC. Epidemics noted throughout middle ages New pathogenic strains continually arise. High mortality in the elderly, very young chronically ill & immunocompromised.

Assembly and Exit of Virions

Formation of the RNA dimer commences by base pairing between complementary sequences in the loop of the dimerization initiation site (DIS) near the 5 end of each RNA. Formation of a 'kissing-loop complex' is believed to stabilize the dimer. The basic NC domains with their zinc fingers bind to the virus genome, initially to a domain known as ψ, which is the main part of the packaging signal. The CA domains bind the host protein cyclophilin A, while p6 domains bind the virus protein Vpr.

What a function does the Tat protein of HIV confers during HIV infection?

Functions as a transcription factor to speed up the transcription of viral genome ( the provirus)

Late Gene Expression

Gag and Gag-Pol are translated from unspliced transcripts, with Gag-Pol translated when a ribosomal frameshift takes place( roughly 5% of occasions when a ribosome traverses the slippery sequence UUUUUUA). This sequence together with a downstream secondary structure, causes the ribosome to slip from reading frame 1 to reading frame 3. The Vif, Vpr, Vpu and Env proteins are translated from singly spliced transcripts. Vpu and Env are translated (leaky scanning) in the rough endoplasmic reticulum from a bicistronic transcript .

Retrovirus Proteins

Genomic RNA associated proteins: Nucleocapsid protein (NC), which coats the RNA. Integrase protein (IN) Reverse transcriptase(RT) Both In and RT are present in much smaller amounts Ribonuclease H (RNase H) Protease Capsid protein (CA). The shape of the capsid: spherical, cylindrical or conical Matrix protein (MA). Envelope associated proteins Transmembrane protein (TM); Glycosylated surface protein (SU). In most retroviruses the bonds between the TM and SU proteins are non-covalent

Foot and Mouth Disease virus

Has a much wider host range than other picornaviruses; it infects mammals such as cattle, sheep, goats and pigs, causing lesions on the feet and in the mouth

HIV Virion

Has the general characteristics of retroviruses except that the capsid is cone shaped, which has a diameter of 40-60 nm at the wide end and about 20 nm at the narrow end. Generally, one capsid per virion.

HIV-1 Variability

High error rate in genome replication results in the variability of HIV-1 as manifested in antigens, host cell range and resistance to drugs. The surface protein gp120 is one of the most variable. There are five domains of gp120 that are especially variable. Interestingly, the Nef protein, which is not on the virion surface, is also very highly variable. Transmission of HIV-1 to a new host is almost always associated with R5 strains, and these predominate during the acute and asymptomatic phases of infection. In about 50% of infected individuals there is evolution towards X4 and R5X4 strains as AIDS develops The presence of anti-retroviral drugs in the body of an infected host exerts an evolutionary pressure which forces drug-resistant variants to emerge more quickly in order to survive.

Reverse Transcriptase (RT)

Highly conserved Heterodimer Major class of anti-HIV drugs are RT Inhibitors

Retroviruses

Hosts: mammals birds other vertebrate animals Diseases: immunodeficiency diseases leukaemias solid tumours *Class VI reverse-transcribing virus replication replicases: DNA-dependent RNA polymerase produced by host and RNA-dependent DNA polymerase produced by virus

Nomenclature of Reovirus

Icosahedral viruses with dsRNA genomes isolated from the respiratory and enteric tracts of humans and animals, and with which no disease could be associated (orphan), became known as reoviruses

Natural Hosts and Pathogenesis of rhabdoviruses

Important pathogens of animals and plants Many rhabdoviruses have very wide host ranges and replicate in the cells of diverse types of host, especially the so-called 'plant' rhabdoviruses, which replicate in their insect vectors as well as in their plant hosts

Prevention and Treatment of Hepatitis A Infections

Interrupt fecal-oral spread Avoid contaminated water or food (undercooked shell fish) Frequent and proper handwashing Prophylaxis with immune globulin before or early in incubation (< 2wks post exposure) is 80 - 90% effective against hepatitis development. The inactivated vaccine is now available for those at risk

9.What's the major function of rotavirus VP3 protein? Is it considered as structure protein?

It has guanylyl transferase and methyl transferase activities, which help add cap to the 5' end of early transcripts It is a structural protein.

5.How does rotovirus with intact VP4 proteins (the surface spike) enter host cells?

It would enter through endocytosis.

2. Which of the following rhabdovirus proteins possesses multiple enzyme activities? a. L protein b. M protein c. N protein d. G protein

L protein

Late Transcription and Translation

Late transcription occurs within the double-layered particles with the late transcripts produced uncapped by viral RNA dependent RNA polymerase (VP1) The translation machinery of the cell undergoes changes such that it selects uncapped transcripts in preference to capped transcripts. Hence, translation of cell proteins is shut down while translation of virus proteins continues The mRNA species are not made in equal numbers, although there are equal numbers of each of the 11 dsRNAs. Virus also uses several mechanisms to control the quantity of each protein produced. Virus proteins are produced at different anounts. e.g. a large amount of the main capsid proteins (VP6) versus relatively small amount of VP1s

6.Where does rotavirus assembly occur?

Lumen of rough ER

Inhibition of Host Gene Expression in rhabdovirus

M protein inhibits transcription by all three host RNA polymerases and blocks intracellular transport of cell RNAs and proteins Direct effects: Inhibition of interferon synthesis (which is induced in response to viral infection) thus the host antiviral immunity More resource available for viral RNA and protein synthesis

The HIV Genome

Main HIV genes: gag: group-specific antigen (encodes matrix, capsid, p2, nucleocapsid, p1 and p6) pol: polymerase (encodes p6*, protease, reverse transcriptase, RNase H, integrase) env envelope Auxiliary genes nef: negative regulatory f actor rev: regulator of expression of virion proteins tat: transactivator of transcription vif: virion infectivity f actor vpr: viral protein R vpu: viral protein U

Poliovirus

Majority of poliovirus infections are relatively harmless infections of the oropharynx and the gut Serious disease occurs only after other tissues become infected, resulting in viraemia (virus in the blood) and spread of infection to the central nervous system. This is a very rare event in babies, who still have anti-poliovirus antibodies acquired from their mothers Less poliovirus in the human environment means little/no antibodies, then infection of the central nervous system is more likely Polio was a disease primarily associated with improving standards of hygiene and sanitation !

HIV Resistant Individuals

Most HIV-1 strains use CCR5 and are known as R5 strains. Individuals who contain homozygous mutation in the CCR5 will be highly resistant to HIV infection. Heterozygous individuals have increased resistance. The mutation is found mainly in Europeans. HIV-1 strains that use CXCR4 as a co-receptor are known as X4 strains, and strains that use CCR5 and CXCR4 as co-receptor are known as R5X4 Strains R5 strains do not infect naive T cells, but all three strains infect memory T cells.

Plant Infecting Reovirus

Most of the plant infecting reoviruses are transmitted between plants by insect vectors The viruses replicate in both the plant and the insect, generally causing disease in the plant, but little or no harm to the infected insect

Rhabdovirus translation

N,P, M and L proteins are translated on free ribosomes, whereas G protein is translated in the rough ER The P and G proteins undergo post-translational modification. One-sixth of the residues in VSV P protein are serine and threonine, and many of these are phosphorylated. The first step of phosphorylation performed by a cell kinase and the second by the kinase activity of the L protein. After phosphorylation, trimers of P are formed. Glycosylation of G protein commences in the rough endoplasmic reticulum, where core monosaccharides are added, and is completed in the Golgi complex.

Other HIV proteins

Nef, Vpr, Vif, p1, p2, p6 and p6*. Major histocompatibility complex class II proteins (associated with the envelope) Cyclophilin A (associated with the capsid).

Do rhabdoviruses require a primer for their genome replication?

No they do not.

6.Do picornaviruses always carry their replicase (RNA-dependent RNA polymerase) inside mature viral particles?

No. they make it after cell entry

The Rotavirus Proteins

Numbered in order of their sizes The three largest proteins are found towards the centre of the virion; 12 copies of VP1 and VP3 are associated with the genome within the inner capsid layer (VP2) VP1 is the RNA-dependent RNA polymerase while VP3 has guanylyl transferase and methyl transferase activities. They are considered structure proteins. There is one copy each of VP1 and VP3 attached to the inner capsid layer at each of the 12 vertices of the icosahedron

Activation of Viral Protein Translation

Once the virus genome is free in the cytoplasm the VPg is removed from the 5 end by a cell enzyme and viral protein translation will ensue.

Poliovirus pt.2

One of the first viruses to be propagated in cell culture ( by Enders, Weller and Robbins, 1949) and also one of the first to be plaque purified (by Dulbecco and Vogt, 1954) § First inactivated vaccine used (Salk 1950's) § First picornavirus to be sequenced § First infectious cDNA clone of an animal virus § First picornavirus structure to be solved Most picornaviruses grow readily in cell culture, are easy to purify and are stable, making them popular viruses for laboratory studies

Assembly and Exit of Rhabdovirus

Only coated minus strands are selected to form virions, because of the presence of a packaging signal at the 5' end of the minus strand The M protein condenses the nucleocapsid into a tightly coiled helix and links the nucleocapsid with a region of the plasma membrane into which copies of the G protein have been inserted. The M protein also has a late (L) domain that binds cell proteins involved in the budding process Virions bud from these regions of the plasma membrane, acquiring their envelopes in the process Nucleorhabdovirus replicate in the nucleus of their plant and insect hosts, with virions budding through the inner nuclear membrane

Describe how HIV genome is replicated

Please refer to lecture slides for details. Pay attention to the enzymes used and primers used for plus and minus strand DNA synthesis. Reverse transcription takes place within the reverse transcription complexThe primer for synthesis of the (−) DNA is the tRNA bound to the genome, while the primer for synthesis of the (+) DNA is a polypurine tract (PPT) in the virus genome, which becomes accessible as a result of hydrolysis of the genome RNA from the 3 end by the RNase HThe DNA that results from reverse transcription (the provirus) is longer than the RNA genome. Each of the termini has the sequence U3-R-U5, known as a long terminal repeat (LTR)-Unlike simple retrovirus HIV will continue to replicate and form a central DNA flap

The Primer for Viral Genome Replication

Poliovirus RNA polymerase uridylylates the small protein VPg, at the hydroxyl group of a tyrosine residue, which is then used as primer for synthesis of both (+) and (−) strand viral RNAs picornavirus genome is the only material required to initiate replication

Poliovirus Pathogenesis and Vaccine

Poliovirus infection of the central nervous system may result in meningitis (fever, headache and neck stiffness), from which most patients recover completely) When virus attack the motor neurons of the spinal cord or the brain stem, it can cause encephalitis and/or paralytic poliomyelitis shown as paralysis of limbs and/or breathing muscles Inactivated vaccines and live attenuated Vaccines have been developed and have proved very effective in preventing polio Polio has been eradicated from many parts of the world and there is hope that the disease will soon be completely eradicated Poliomyelitis is now present only in North & Central Africa, India, & Pakistan. Rapid identification & surveillance are very important to prevent import and spread of the virus.

8.What are the major translation Strategies used by Plus-stranded RNA Viruses in Class IV.

Polyprotein, segmented genome and subgenomic mRNA

Translation Strategies of Plus-stranded RNA Viruses

Polyprotein. All the genetic information is encoded in one ORF; this is translated to produce a polyprotein which is cleaved to produce the individual virus proteins. • Subgenomic mRNAs. The genome has two or more ORFs. The RNA-dependent RNA polymerase is encoded by the ORF at the 5 end of the genome so that it can be translated from the infecting genome. The other ORF(s) are transcribed into subgenomic mRNA(s) that have the same 3 end as the genome • Segmented genome. There is one ORF in each RNA segment ((open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons)

Transportation of Pre-integration Complex

Pre-integration complexes, which contains host proteins as well as virus proteins, are transported to the nucleus along microtubules. The pre-integration complex of HIV, unlike those of most retroviruses, can enter an intact nucleus, such as that of a resting T cell or a macrophage, likely transported through a nuclear pore. Nuclear localization signals have been identified in MA, Vpr and IN. Integration of HIV provirus in resting memory CD4 T cell may result in a latent infection, allowing the virus to survive in individuals receiving anti-retroviral drug therapy.

Hepatitis A Virus

Prevalent in developing countries with poor sanitation Most infants and young children do not develop symptoms (or very mild) and leads to life-long immunity When adults become infected about 75% develop jaundice; severe hepatitis is a rare complication, which can be fatal

Endogenous Retroviruses (ERV)

Primarily in vertebrate animal genomes (Retroviruses only attack vertebrates) The genomes of most of these ERV are defective Sequencing the human genome has revealed the presence of almost 100 000 human ERV (HERV) sequences, and ERVs have been found in the genomes of other species Some ERVs are closely related to normal retroviruses (exogenous retroviruses). It is thus highly likely that ERVs originated as a result of exogenous retroviruses infecting germ line cells (sperm and/or egg). Over time ERVs have copied themselves to other locations in the genome, giving rise to families of related ERV elements.

Transcription/Genome Replication of poliovirus

RNA synthesis occurs in replication complexes that contain cell proteins, as well as virus proteins and RNA The replication complexes are associated with the cytoplasmic surfaces of membranous vesicles derived mainly from the endoplasmic reticulum Each vesicle is bounded by two lipid bilayers. All known class IV viruses of eukaryotes replicate their RNA in similar membrane associated complexes

The enzyme activities of rhabdovirus L protein

RNA-dependent RNA polymerase Methyltransferase Guanylyltransferase Poly A synthase activity Kinase

Describe all enzymatic activities of the L protein produced by rhabdoviruses.

RNA‐dependent RNA polymerase Methyl transferase Guanylyl transferase Poly A synthase activity Kinase

3.What's the major function of rotavirus VP1 protein? Is it considered as structure protein?

RNA‐dependent RNA polymerase. It is a structure protein

Rabies Virus Vaccine

Rabies virus vaccines have been developed to provide protection to humans, domestic animals such as dogs and wild animals, which are at risk from rabies virus infection The first vaccine to be used was an attenuated vaccine, but more recent vaccines have contained a recombinant vaccinia virus that expresses the rabies virus G protein. Vaccination of wild mammals has been very successful in bringing rabies under control in a number of countries Many viruses related to rabies virus have been found in bats around the world, and have been classified in the genus Lyssavirus along with the original rabies strains. There are occasional cases of human rabies resulting from bites from infected bats

Generation of virulent virus through genome recombination

Recombination may occur in between vaccine strains and between vaccine strains and wildtype strains There have been a number of documented cases where recombinants have caused polio

Prevention and Treatment (of rotaviruses?)

Rehydrating the patient with a solution of salts and sugar. Gatorade and other sports drinks are not recommended as they contain high amounts of sugars that can further irritate the intestinal tract. Two vaccines are currently being given with success: RotaTeq and Rotarix.

Reverse Transcription

Reverse transcription takes place within the reverse transcription complex The primer for synthesis of the (−) DNA is the tRNA bound to the genome, while the primer for synthesis of the (+) DNA is a polypurine tract (PPT) in the virus genome, which becomes accessible as a result of hydrolysis of the genome RNA from the 3 end by the RNase H The DNA that results from reverse transcription (the provirus) is longer than the RNA genome. Each of the termini has the sequence U3-R-U5, known as a long terminal repeat (LTR) Very Error-prone 1 error in 104 bases; = 1 per replication. Namely ~100 Mutations will be introduced into the progeny genome after each replication cycle.

Major Nonsegmented Negative Strand Virus Families

Rhabdoviridae - 70 X 250 nm bacilliform particles. - Membrane spikes & Helical Nucleocapsids. - 1 segment, 11-14 kb ssRNA genomes. > 175 members in the family. - Members infect animals, fish & plants. Vesicular Stomatitis & Rabies virus. Filoviridae - 80 x 800 nm or longer filaments. - Long branched or U shaped nucleocapsids. - 1 ssRNA 13 kb genome. - Two characterized members: Marburg & Ebola. Paramyxoviridae - Pleomorphic, 150 to 250 nm diameter. - Prominent glycoprotein spike. - Filamentous helical nucleocapsids - 15 kb ssRNA genome. - Human & animal pathogens: Measles, Mumps & Canine Distemper.

Diarrhea Causing Agents in World

Rotaviruses, ranking sixth among the worlds global killers, are equal opportunity viruses. Prevalence is about the same in all children. Good sanitation does not reduce incidence

1.What are the structure features of reovirus genome ?

Segmented dsRNA -icosahedral symmetry -double stranded RNA genome -60-80 nm diameter

Rescue of ERV Replication

Some ERVs can replicate when the missing functions is supplied by another ERV or an exogenous retrovirus. Some ERVs do not replicate in cells of the species in which they occur, but are able to replicate in the cells of other species; e.g., some mouse ERVs and some pig ERVs can replicate in human cells. (Potential problem in reality: there may be a risk of transmitting retroviruses from pigs into humans if pigs are used as sources of cells, tissues and organs because of shortages of their human counterparts for transplant purposes.) There are also some ERVs that are not defective; they have an intact genome (gag, pol and env genes) and can initiate a productive infection.

Hosts of mononegavirale

Some Negative-strand RNA viruses have Vertebrate, Invertebrate and Plant Hosts. Vertebrate Negative-strand viruses are more diverse than those of Plants and Invertebrates. All plant-infecting negative strand RNA viruses also infect their insect vectors.

Early Events of Rotavirus Replication (cont.)

Some of the virus proteins undergo co- and post-translational modifications for function: VP2 and VP3 are myristylated while NSP5 is phosphorylated and O-glycosylated The virus proteins accumulate within the cytoplasm in discrete regions known as viroplasms NSP2 and NSP5 play roles in the formation of viroplasms: reducing the expression of NSP5 by RNA interference resulted in fewer and smaller viroplasms In the viroplasms virion cores are assembled from VP1, VP2 and VP3

Retrovirus Assembly

Some retroviruses form immature particles in the cytoplasm that are then transported to the plasma membrane Most retroviruses assemble their components on the inner surface of the plasma membrane, with the N termini of the Gag and Gag-Pol proteins become anchored to the plasma membrane by the myristyl groups, and the association is stabilized through electrostatic interactions between positive charges in the MA domains and negatively charged phosphate groups in the membrane. The MA domains also bind to the cytoplasmic tails of TM proteins in the membrane. The NC domains of Gag and Gag-Pol bind the polyproteins to the virus RNA and mediate the formation of the genome dimer. The proteins bind first to a packaging signal near the 5 end of each RNA molecule, and a tRNA binds to the PBS. The RNA then becomes coated with many copies of Gag and a few copies of Gag-Pol.

Inhibition of Host Gene Expression

Soon after infection, the expression of host cell genes is inhibited. As is shown in poliovirus-infected cells all three host RNA polymerases are rapidly inhibited, thus all transcription events are terminated Translation from pre-existing cell mRNAs is inhibited as a result of the cleavage of a translation initiation factor (4F) by the virus protease 2A Virus protein synthesis is unaffected as it is initiated by a cap-independent mechanism

5' End Structure of HIV Genome

TAR: trans-acting response element Poly-A: polyadenylation signal PBS: primer-binding site DIS: dimerization initiation site (involved in formation of kissing loop complex) SD: splice donor site Psi(ψ): main part of the packaging signal AUG: start codon of the gag gene

Picornavirus Assembly and Exit

The 60 copies of VP0 are cleaved into VP4 and VP2 when each procapsid acquires a copy of the virus genome, with VPg still attached at the 5 end Lysis of the cell releases the virions; approximately 105 virions are produced in each poliovirus-infected cell

HIV Proteins

The NC protein of HIV-1 is highly basic and has zinc fingers. The TM and SU proteins are named gp41 and gp120 (gp = glycoprotein); gp120 is heavily glycosylated. The C terminus of gp41 is inside the virion, where it is bound to the MA protein. Each spike is a gp41-gp120 trimer and there is an average of 14 spikes on each virion. The equivalent glycoproteins in HIV-2 (gp38 and gp130) are unrelated to those of HIV-1, whereas most of the internal proteins of the two viruses are related.

Functional Roles of Nef, Tat and Rev

The Nef protein: alter the endosome trafficking pathway, reducing the expression of CD4 and MHC class I and II proteins, thus shielding HIV-infected cells from immune surveillance. The Tat (trans-activator of transcription) protein plays an important role in enhancing transcription. Binding to TAR increases the processivity of the RNA polymerase along the proviral template. Tat is a transcription factor that binds to RNA, but not to DNA. Rev (Regulator of expression of virion proteins) protein, has a nuclear localization signal. Rev binds to the Rev response element (RRE) in the virus RNA and causes a shift from early to late protein synthesis. The RRE is present in the unspliced and singly spliced transcripts, but is absent from the multiply spliced transcripts. The late genes are translated from genome-length and singly spliced transcripts, but these mRNAs are not transported from the nucleus until they have bound multiple copies of Rev.

HIV Genome

The auxiliary genes: control virus gene expression, transport virus components within the cell and modify the host's immune response. Some of their products have multiple roles. All three reading frames are used with extensive overlapping; e.g., part of vpu in frame 2 overlaps env in frame 3. The sequences for tat and rev are split, the functional sequences being formed when the transcripts are spliced.

The Capsid of Human Rhinovirus 14

The canyons, lined by the C termini of VP1 and VP3 molecules, contain the virus attachment sites

ENV Protein Translation and Post-Translational Modifications

The env gene is translated from spliced mRNAs in the rough endoplasmic reticulum, where glycosylation commences. The Env protein molecules are then transported to the Golgi complex, where they are cleaved by a host protease into SU and TM molecules. The two cleavage products remain in close association, and after further glycosylation they are transported to the plasma membrane.

GAG and POL Protein Translation

The gag and pol genes are translated from genome-length mRNAs into Gag and Gag-Pol polyproteins. Approximately 95 per cent of ribosomes terminates translation after the synthesis of Gag, while the other ribosomes continue translation to synthesize Gag-Pol.

Translation and Post-translational Modifications for poliovirus

The genome encodes a single polyprotein, which undergoes a series of cleavages to give rise to all the structural and non-structural proteins

Rhabdovirus Genome Organization

The genomes of all rhabdoviruses encode at least five proteins. Many rhabdoviruses encode one or more proteins in addition to these. Short intergenic sequences exist in between these genes

5.Describe the terminal structure features (such as cap and polyA tail) of retrovirus genome and mRNA transcripts

The genome‐length transcripts serve as both viral genome and mRNA. Both genomic RNAs and mRNAs carry cap at 5' end and polyA tails at 3' end.

Retrovirus Assembly - Late Stages

The immature virion acquires its envelope by budding from the cell surface. Multiple copies of Gag and Gag-Pol are arranged radially with their N termini facing outward and their C termini inward. The late (L) domains of Gag bind host cell factors that are involved in the budding process. During and/or after the budding process the Gag and Gag-Pol polyproteins are cleaved by the virus protease. The cleavage products of Gag form the matrix, the capsid and the protein component of the nucleocapsid, while the Pol cleavage products are the virion enzymes.

Rhabdovirus Genome Replication and Secondary Transcription

The initiation of RNA synthesis does not require a primer. All Rhabdovirus, except nucleorhabdovirus, replicate in cytoplasm There are termination signals for RdRP at each intergenic sequence of the template and at the end of the L gene During genome replication, however, the enzyme must remain associated with the template to produce genome-length (+) RNA. The newly synthesized (+) RNA quickly becomes coated with N protein (become protected from ribonucleases), whereas the mRNAs are not coated. This is true for all minus-strand RNA viruses Possible mechanisms involved in synthesis of mRNA and genome length (+) RNA: "Transcriptase" versus "replicase" (-) genome length RNA : (+) genome length RNA=4-10:1

HIV Attachment and Entry

The interaction of gp120 with the receptor and co-receptor results in a dramatic re-arrangement of gp41,which proceeds to fuse the membranes of the virion and the cell. The contents of the virion envelope released into the cytoplasm will develop into the reverse transcription complex, which contains the MA, Vpr, RT and IN proteins, as well as the virus genome.

Ribosomal Frameshifting for Gag and Pol Protein Translation

The majority of retroviruses using ribosomal frameshift mechanism to ensure the correct proportions of the Gag and Pol proteins gag and pol are in different reading frames and there is a −1 shift in reading frame before the gag-pol junction in about five per cent of translations. This mechanism is used by HIV-1. The Gag and Gag-Pol proteins of most retroviruses are myristylated at their N termini.

Rhinoviruses

The most common agents causing upper respiratory tract infections in humans Most children have had at least one rhinovirus infection by the age of 2 years, and in adults rhinoviruses account for about 50% of common colds Replicate in the epithelium of the upper respiratory tract, where the temperature is around 33-35C, optimum temperature for some human rhinoviruses to replicate. Many human rhinoviruses are able to replicate at 37 ◦C and some of these are probably responsible for disease in the lower respiratory tract Why can't we protect against the cold? more than 100 rhinoviruses!

Rotavirus: Summary of structure features

The most important agents of gastroenteritis in humans and animals Spherical virions of icosahedral symmetry, with structures resembling the spokes of a wheel, therefore named after the Latin word rota (= wheel) Triple-layered capsid with each layer constructed from a distinct virus protein (VP). The inner and middle layers, constructed from VP2 and VP6 respectively, are perforated by channels. The middle layer contains the 'spokes' of the 'wheel' and is the major component of the virion The outer layer is constructed from glycosylated VP7. VP7 is associated with a membrane (ER) within the cell before it is incorporated into the virion VP1 and VP3( both are considered structure proteins) in the core, and VP4 forms 60 spikes at the surface VP1 and VP3 always in mature virus

Retrovirus Assembly-Early Stages

The order of the Gag domains MA-CA-NC is the same as the exterior-to-interior order of the proteins in the virion

Early Events of Rotavirus Replication

The outer layer of the virion is removed, leaving a double-layered particle in which transcription is activated VP1 synthesizes a new copy of the (+) RNA, and a molecule of VP3, which caps the 5 end of the new RNA Since the inner and middle layers are perforated by channels, free nucleosides can enter this double-layered structure for the synthesis of (+) genomic RNAs and early mRNAs, which are extruded from the particles through the channels The transcripts are not polyadenylated (therefore no polyA tail)

Assembly and Exit cont

The p6 domain functions as the late (L) domain, responsible for the release of the budding virion from the host cell. This domain contains the sequence proline-threonine-alanine-proline (PTAP), which can bind cell proteins important in the pinching off process. Vif proteins bind cell enzymes (APOBEC3F and APOBEC3G) and induce their degradation. Gag-Pol proteins form dimers, then undergo self-cleavage to form the virus enzymes, including the protease, which is a dimer. The protease then cleaves the Gag polyproteins into the constituents of the mature virion.

Picornavirus Genome

The picornavirus genome is composed of a 7-8 kb ssRNA Covalently linked to the 5 end of the RNA is a small (2-3 kD) protein known as VPg (virus protein, genome linked). The covalent link is via the -OH group of a tyrosine residue at position 3 of VPg The 3 end of the RNA is polyadenylated.

Transcription/Genome Replication

The poly(A) sequence at the 3 end of the plus strand is transcribed from a poly(U) sequence at the 5 end of the minus strand For poliovirus, (+) RNA :(-)RNA = 50:1

VPg Priming Model of Poliovirus RNA Synthesis

The precursor of VPg, 3AB, participates in the initiation reaction. The 3AB polypeptide binds to the Endoplasmic Reticulum and acts in replication complexes as a VPg donor. Uridylation of membrane-bound 3AB is carried out by 3D pol and is dependent on the presence of an RNA template and occurs in vesicles formed from the ER. VPg-pUpU is then cleaved from 3A by the viral protease 3C and serves as an elongation primer. The released 3Dpol primes minus strand RNA synthesis at the 3' end of the plus strand with VPg-pUpU as a primer.

HIV Pathogenesis

The profound immunosuppression seen in AIDS is due to the depletion of T4 helper lymphocytes. In the immediate period following exposure, HIV is present at a high level in the blood (as detected by HIV Antigen and HIV-RNA assays). It then settles down to a certain low level (set-point) during the incubation period. During the incubation period, there is a massive turnover of CD4 cells, whereby CD4 cells killed by HIV are replaced efficiently. Eventually, the immune system succumbs and AIDS develop when killed CD4 cells can no longer be replaced (witnessed by high HIV-RNA, HIV-antigen, and low CD4 counts).

Integration of the Provirus

The provirus, still associated with some virion protein, is transported to the nucleus as a pre-integration complex. For most retroviruses this can occur only if the cell goes into mitosis that results in breakdown of the nuclear membranes HIV and related viruses, however, can productively infect resting cells, as the pre-integration complexes of these viruses are able to enter intact nuclei. Integrase cuts the DNA of a cell chromosome and seals the provirus into the gap. The integrated provirus genes may be expressed immediately, or there may be little or no expression of viral genes, in which case a latent infection has been initiated. If a latently infected cell divides, the provirus is copied along with the cell genome and each of the daughter cells has a copy of the provirus

HIV Reverse Transcription

The reverse transcription complex associates rapidly with microtubules. Reverse transcription is primed by tRNA lys-3. Most proviruses of retroviruses are entirely dsDNA, whereas those of HIV and other lentiviruses have a short triple-stranded sequence known as a central DNA flap, which plays a vital role in the early stages of infection.

The Rotavirus Genome

The rotavirus genome is made of 11 dsRNA segments Each RNA segment encodes one protein, with the exception of one segment, which encodes two proteins Altogether 12 proteins are encoded: six structural proteins (VP) and six non-structural proteins (NSP)

Background/Current Situation of HIV

The simian immunodeficiency viruses (SIV) do not harm their natural primate hosts. Both viruses emerged in the late 20th century. their infection damages the immune system, thus called acquired immune deficiency syndrome (AIDS), leaving the body susceptible to infection with a wide range of bacteria, viruses, fungi and protozoa. It is HIV-1 that is largely responsible for the AIDS pandemic, while HIV-2 is mainly restricted to West Africa. There are approximately 5 million new HIV infections, and approximately 3 million deaths from AIDS each year, the fourth biggest cause of mortality in the world. So far there has been success in developing anti-HIV drugs.

Transcription and Genome Replication of retroviruses

The two LTRs of the provirus have identical sequences, but transcription is initiated in one and terminated in the other. There is a polyadenylation signal in the R region and transcription terminates at the R-U5 junction. Each transcript is capped and polyadenylated. Some transcripts will function as mRNA and a proportion of these become spliced; others will become the genomes of progeny virions. Transcription of viral RNA from the proviral DNA uses the cellular transcriptional machinery (host DNA-dependent RNA polymerase II) (A long terminal repeat (LTR) is a pair of identical sequences of DNA)

Virion Assembly cont.

The vesicle membrane forms a temporary 'envelope' which contains VP7 Cleavage of VP7 molecules releases them from the membrane to build up the outer layer of the virion and VP4 is added to form the virion spikes Virions are released from the cell either by lysis or by exocytosis.

Attachment and Entry of retroviruses

The virion binds to cell receptors via the virus attachment site on the SU protein. This interaction causes a conformational change in the TM protein that allows a hydrophobic fusion sequence to fuse the virion membrane and a cell membrane. Most retroviruses fuse their membrane with the plasma membrane of the cell, though some are endocytosed and fuse their membrane with an endosome membrane. The structure that is released into the cytoplasm loses some proteins and a reverse transcription complex is formed. *genome requires primer: primer is tRNA

Other Important (-) RNA Virus

Three of the world's major human pathogens: influenza virus, measles virus and respiratory syncytial virus (RSV) Respiratory syncytial virus: Highly contiguous, causes severe diseases on young and old people Measles virus: Mild symptom on adults but severe diseases on infants and kids. Immunosuppression leads to severe diseases resulted from co-infection of other pathogens

8.How does rotavirus exit host cells?

Through cell lysis or exocytosis.

Describe how HIV Gag-Pol polyprotein is produced.

Through ribosomal frameshift

Early Gene Expression

Transcription of HIV genes is initiated after cell transcription factors bind to promoter and enhancer sequences in the U3 region of the upstream LTR. Transcription is terminated in the downstream LTR; the polyadenylation signal AATAAA is in the R region and transcripts are polyadenylated at the R-U5 junction. Many of the genome-length transcripts are spliced to produce three size classes of virus transcripts. The largest RNAs are genome length (about 9.3 kb), while the other two size classes are each made up of a number of mRNA species that have undergone splicing; mRNAs that have been spliced once are around 4.5 kb, while mRNAs that have undergone two or more splicing events ('multiply spliced' transcripts) are around 2 kb. The virus genome has a number of splice donor sites and acceptor sites; these enable splicing events that result in more than 30 mRNA species. Early in infection most of the primary transcripts are multiply spliced and these RNAs are translated into the Nef, Tat and Rev proteins.

Rhabdovirus Transcription

Transcription of the virus genome begins right after the RNA and its associated proteins (N, P and L) are free in the cytoplasm A plus-strand leader RNA, the function of which is uncertain, and five mRNAs are synthesized. Transcription is carried out by an RNA-dependent RNA polymerase activity that resides, along with four other enzyme activities, in the L protein The polymerase is active only when L is complexed with P protein in the ratio 1L:3P The L protein also have enzyme activities that cap and polyadenylate the mRNAs Rhabdovirus controls its gene expression by controlling the relative quantities of transcripts synthesized

Cell Entry of Picornavirus

Transfer of the RNA from the virion into the cytoplasm, leaving the capsid at the cell surface Endocytosis, followed by either release of genomic RNA from the capsid after disruption of the endosome membrane, or release of genomic RNA from the capsid then transport across the endosome membrane

Site-specific cleavage by Trypsin and chymotrypsin

Trypsin: cleaves the peptide bonds after (on the C-terminal side of) the basic amino acids lysine and arginine Chymotrypsin: prefers cleaving after large hydrophobic amino acids such as phenylalanine, tyrosine, tryptophan etc.

Retrovirus Virion Structure

Two copies of the RNA genome, hence a diploid, present as a dimer, forming a 'kissingloop complex'. Also contains host RNAs that were packaged during assembly. A molecule of transfer RNA (tRNA) bound to each copy of the virus RNA through base pairing. The sequence in the virus RNA that binds a tRNA is known as the primer binding site (PBS) (so requires primer) Each retrovirus binds a specific tRNA

"Suppressor tRNA" for pol Gene Translation

Used by Murine leukaemia virus (MLV). The mechanism involves reading through a stop codon at the end of gag. A 'suppressor tRNA' incorporates an amino acid at the stop codon and translation continues into pol, which is in the same reading frame as gag.

4.Which viral protein is released when a poliovirus is bound to the cell receptors?

VP4

Virion Assembly

VP7 and NSP4 are synthesized and N-glycosylated in the rough endoplasmic reticulum, where they remain localized in the membrane NSP4 has binding sites for both VP4 and double-layered particles and is required for initiation of immature virion binding to ER After binding these components the immature virion buds through the membrane into a vesicle within the endoplasmic reticulum

VSV Transmission

VSV has a very wide host range. As well as infecting domestic livestock there is evidence of infection in wild animals including bats, deer and monkeys, as evidenced the presence of neutralizing antibodies to VSV in these animals VSV has been isolated from a number of insect species, including mosquitoes, sand flies and black flies. Its natural cycle is unknown, but it is possible that it is transmitted between mammals by one or more of these types of insect In the laboratory VSV can replicate in cell cultures derived from mammals, birds, fish, insects and nematode worms. Much of our understanding of rhabdovirus structure and replication comes from studies with VSV, a much safer rhabdovirus compared to rabies virus Recombinant vesicular stomatitis virus-Zaire Ebola virus vaccine 2016 Altogether three species of VSV are documented so far

Reovirus at A Glance

Very broad host range: mammals, birds, fish, insects, plants and fungi Disease: gastroenteritis in humans and animals Virion: • Icosahedral symmetry • 60-80 nm diameter • Genome: double-stranded RNA 10-12 segments 18-32 kbp

Vesicular Stomatitis Virus

Vesicle = blister Stomatitis = inflammation of mucous membrane in the mouth Natural hosts of VSV: cattle, horses, sheep and pigs Disease symptoms: lesions on the feet and in the mouth similar to those in foot and mouth disease The disease can result in significant economic damage due to decreased milk and meat production, and the imposition of quarantines and trade barriers. Vesicular stomatitis is endemic in the tropics and there are cyclic epidemics in some temperate areas

5.Describe the structure features of a picornavirus genome.

Vpg covalently linked to the 5'end. Poly A tail at the 3' end. The genome contains a single open reading frame which encodes a polyprotein

4.During retrovirus genome replication, the reverse transcription occurs in cytoplasm, right?

Yes

Can RNA dependent RNA polymerase or its activity be found in mature virion of rhabdoviruses?

Yes

9.Do picornaviruses need a primer to replicate their genomes?

Yes, it is the VPg protein.

2.Can poliovirus genomic RNA alone be used to initiate viral infection?

Yes.

Endopeptidases

break internal peptide bonds and initially produce peptide fragments. Examples: trypsin, chymotrypsin, pepsin and furin

How do rhabdoviruses enter host cell?

endocytosis

Types of proteases:

endopeptidases and exopeptidases

Retrovirus Genome Organization

gag: group-specific antigen (internal structural protein) pol: polymerase (enzymes) env:envelope proteins PBS: primer-binding site R: direct repeat sequence U3: unique sequence at 3 end of genome U5: unique sequence at 5 end of genome RNA has a 5' cap and a 3' poly (A) tail Looks like eukaryotic mRNA ( BUT doesn't serve as mRNA immediately after infection! No chance to be bound by ribosomes). Reverse transcription occurs in the particle right after infection

2.What's the major disease caused by rotavirus infection?

gastroenteritis in humans and animal

2.The transcription of retrovirus genome is carried out by which cellular enzyme?

host DNA-dependent RNA polymerase II

The transcription of HIV is carried out by which cellular enzyme?

host DNA-dependent RNA polymerase II

peptides undergo _________ to become amino acids

hydrolysis

Describe the terminal structure features (cap and polyA tail) of rhabdovirus genomic RNA and mRNAs.

mRNAs contains 5' cap and 3' polyA tail. These structures are not present on viral genome

Rhabdoviridae dieases

rabies vesicular stomatitis yellow dwarf of potato (potato yellow dwarf virus)

Describe how the rev protein of HIV regulate HIV gene expression

rev carries both nuclear localization and nuclear export signal and thus is able function as a shuttle to export genome‐length and singly spliced mRNAs to cytoplasm for translation.

Example Genera in the Family Reoviridae

rotavirus orthoreovirus

Picornaviruses:Hosts and Diseases Caused

small RNA virus Hosts: Mammals Birds Diseases caused: Common cold Polio Hepatitis A Foot and mouth disease (first animal virus described)

Exopeptidases

start at the end of a polypeptide chain and sequentially remove one or two amino acids at a time

Cell Entry of Poliovirus into Cells

v During interactions of poliovirus with its receptor major conformational rearrangements occur in the virus particle. vThe particles lose VP4 and the hydrophobic N-terminus of VP1 is displaced to the virion surface vN-termini of VP1 forms a pore in the cell mebrane through which the RNA is released into the cytosol. v Some evidence suggests that poliovirus particles may undergo endocytosis in some cell types.

Negative strand RNA Replication

v Nucleocapsids have RNA-dependent-RNA polymerase activity v v Polymerase transcribes the Viral genomic RNA into mRNA progeny and plus-sense antigenomic RNA replicative intermediates v v Antigenomic RNAs are copied into progeny genomic RNAs to produce more RNA or are encapsidated to form progeny virus

Cleavage of Poliovirus Genome by a Cell Enzyme

v Polioviral RNA is linked to the 22 aa VPg via a uridine- tyrosine phosphodiester bond. v During replication, mRNAs are produced by cleavage of this phosphodiester bond by a cellular enzyme to produce viral mRNAs containing a 5' terminal pU.

vaccine for rotaviruses?

yes, 80-100% effective

General Features of Hepatitis A Infection

§ Enterically transmitted (fecal/oral route). § Multiplies in intestional epithelial cells. § Moves into the bloodstream and attacks the Liver. § Four week incubation time. § After 15 to 50 days hepatitis symptoms appear. § Transient hepatitis last at most a few weeks. § Often referred to as "infectious hepatitis". § Only a single serotype exists. § Causes ~40% of acute hepatitis cases. § Vaccine development is successful.

Modes of Rotavirus Entry

§Direct penetration of the virion across the plasma membrane ----Mediated by a hydrophobic region of VP5* which is hidden in the uncleaved VP4, so virions with spike proteins that have not been cleaved are unable to enter by this mechanism § Endocytosis

Picornavirus virion structure:

§icosahedral § 25-30 nm diameter § genome: single-stranded RNA Plus polarity 7-8 kb Covalently linked protein (VPg) Icosahedral capsid made from 60 copies each of four virus proteins (VP1-4) Parts of VP1-3 are at the surface of the virion, while the N termini of VP1-3 and all 60 VP4 molecules are completely internal

Important Features for Class IV Viruses

• Translation occurs before transcription • RNA replication takes place on membranes • The processes of transcription and genome replication are one and the same, resulting in the production of (+) RNA • (+) RNA has three functions: ◦ templates for (-) RNA synthesis ◦ mRNA ◦ genomes of progeny virions.

7.Describe the important features for plus-stranded RNA viruses in Class IV.

• Translation occurs before transcription • RNA replication takes place on membranes • The processes of transcription and genome replication are one and the same, resulting in the production of (+) RNA

Enzymatic Protein: Protease

•10 kd, dimer Protein •Autocatalytic release from Gag-Pol precursor •Aspartyl protease •Exquisite cleavage specificity (In HIV: Ser-Thr-Xaa-Yaa-Phe/Tyr-Pro-Zaa) •Major class of anti-HIV drugs are protease inhibitors.

An Overview of HIV

•Causative agents of acquired immune deficiency syndrome (AIDS), first described in 1981 •Two major types: HIV-1 and HIV-2 •HIV-1 isolated in 1984, and HIV-2 in 1986, HIV-2 shares 40% nucleotide homology with HIV-1 •Belong to the lentivirus genus of the retroviridae

Recorded Influenza Pandemics

•First well-described pandemic occurred in 1580 •Since 1580, there have been 31 known pandemics •In the 20th century: -1918 - Spanish Flu (H1N1) -1957 - Asian Flu (H2N2) -1968 - Hong Kong Flu (H3N2) -2009 - Swine flu H1N1 § Estimated that 9 to 10 % of the world's population contracts influenza each year. § A pandemic of the magnitude of the 1918 Flu would kill between 70 and 140 million people. The world is no better prepared than in 1918 for outbreaks. § Preventing evolution of lethal strains is the key to prevention of pandemics.

Mechanism of Integration

•Integrase binds ends of retroviral DNA and cleaves 2 bases from 3' ends • •Binds host DNA and makes short (4-6 bp) staggered cuts • •Strands transfer to host DNA • •Gaps are repaired • •Integrated provirus has 1 or 2 less bases at LTR end • •No mechanism for excision: present for life of cells • •Inherently mutagenic because of interruption of host genes

Integrase Protein

•Integrates retroviral DNA into host genome • •Endonuclease activity • •Drugs being developed

Common features of dsRNA viruses

•Replicate in cytoplasm (so cannot use host machinery must use their own) •Viral genomes can not be directly translated •Always carry RdRP required for genome replication and mRNA production •Have to use their enzyme to add cap structure to their own mRNAs, if such a structure is required for translation •The genome is a potent trigger of RNAi-based antiviral mechanism

Clinical Features

•Seroconversion illness - seen in 10% of individuals a few weeks after exposure and coincides with seroconversion. •Incubation period - this is the period when the patient is completely asymptomatic and may vary from a few months to a more than 10 years. The median incubation period is 8-10 years. •AIDS-related complex or persistent generalized lymphadenopathy (swollen/enlarged lymph nodes). •Full-blown AIDS.

Prevention of HIV

•The risk of contracting HIV increases with the number of sexual partners. A change in the lifestyle would obviously reduce the risk. •The spread of HIV through blood transfusion and blood products had virtually been eliminated since the introduction of blood donor screening in many countries. •AZT had been shown to be effective in preventing transmission of HIV from the mother to the fetus. The incidence of HIV infection in the baby was reduced by two-thirds. •The management of health care workers exposed to HIV through inoculation accidents is controversial. Anti-viral prophylaxis had been shown to be of some benefit but it is uncertain what is the optimal regimen. •Vaccines are being developed at present but progress is hampered by the high variability of HIV. Since 1987, more than 30 HIV candidate vaccines have been tested in approximately 60 Phase I/II trails, involving more than 10,000 healthy volunteers. A phase III trial involving a recombinant gp120 of HIV subtype B was reported in Feb 2005 to be ineffective in preventing HIV infection.

Historical Events in Retrovirology

•Vallee and Carre (1904) - Filterable virus is cause of "Swamp Fever" of horses (Equine Infectious Anemia Virus,EIAV) •Ellerman and Bang (1908) - chicken leukemia transferred by bacteria-free filtrate (virus) •Rouse (1911) - Rouse sarcoma in chickens (RSV) 1930s - 1970s - RNA tumor virus work in chickens & mice. •Temin and Baltimore (1970) - discovery of Reverse Transcriptase (Nobel laureates) •Bishop and Varmus (1978) - oncogenes are derived from cellular genes involved in growth (src product is cellular phosphokinase) (Nobel laureates) •Gallo (1981) - human retrovirus (HTLV-Human T Cell leukemia Virus) •Barre-Sinoussi (1983) - Human Immunodeficiency Virus isolated and shown to be the cause of AIDS (Nobel laureates)

Treatment for HIV

•Zidovudine (AZT) was the first anti-viral agent shown to have beneficial effect against HIV infection. However, after prolonged use, AZT-resistant strains rapidly appears which limits the effect of AZT. •Combination therapy has now been shown to be effective, especially for trials involving multiple agents including protease inhibitors. (HAART - highly active anti-retroviral therapy). The rationale for this approach is that by combining drugs that are synergistic, non-cross-resistant and no overlapping toxicity, it may be possible to reduce toxicity, improve efficacy and prevent resistance from arising.