Genetic Class III and IV

11-12-09 RNA Viruses: Genetic Class III and IV (+) stranded and dbl stranded genomes 1.
Describe what an arbovirus is. Arboviruses are viruses transmitted by arthropods. The transmission pathway of arboviruses is as follows: Blood sucking infect epithelial cells of the mid-gut viremia infect salivary gland cells virus in saliva virus injection into patient Flaviviruses (includes West Nile, yellow fever, St. Louis encephalitis, Japanese encephalitis) and alphaviruses (including Eastern and Western equine encephalitis viruses) are arboviruses. 2. Describe the individual steps of the life cycle of a positive-stranded RNA virus. Poliovirus is a naked, iscosahedral ss (+) RNA representative of genetic class IV viruses. Structure: spherical capsid with 180 structurally equivalent proteins subunits. T=3 recall C=(T-1)x10+12, where (T-1)x10 is the number of hexamers (so in this case, there are 20 hexamers), while 12 is the constant number of pentamers. Genome: single molecule of RNA with MW of 2x106 and 7500 nucleotides. Life cycle: Adsorption: a temperature independent, reversible process. It is an electrostatic interaction requiring divalent cations, and depends on the ionization of the viral surface as well as the ionization of the poliovirus receptor on the cell surface. Absorbed virus can be washed off a cell surface with high salt or low-pH solutions. Adsorption of poliovirus is also tissue-specific, thus illustrating the concept of tissue tropism. Only certain tissues have on their surface a specific receptor for poliovirus (spinal cord and intestine). Other tissues such as kidney, lung, muscle, heart, and amnion do not have the receptor and are not susceptible to poliovirus. FYI: tissue tropism breaks down in culture, where all tissues seem to start expressing poliovirus receptor; thats why poliovirus vaccine is grown in kidney cells of rhesus monkeys!; demonstrates that all primate cells have the genetic info to produce a poliovirusspecific cell surface receptor, and non-primate cells are therefore not susceptible to poliovirus infection The gene for poliovirus receptor has been isolated and sequenced. It codes for a glycoprotein on the cell surface and has some relationship to the Ig superfamily, but the true function of the receptor has not been identified. Penetration: a temperature-dependent process requiring a VP4 capsid protein, which has a fatty acid attached to the N terminus, possibly mediating some membrane interaction. Uncoating: this is the process by which RNA is liberated from the capsid so that it can direct virus replication (see more about poliovirus replication below).
Uncoating occurs in the last stage of endocytosis. Only 2% of RNA successfully uncoats and initiates infection! Eclipse: refers to the disappearance of viral infectivity early in infection. This disappearance is due to the viral genome being liberated inside the cell, where it is about to begin the direction of viral replication (no progeny has been made yet). Eclipse is considered a hallmark of virus infection. The significance of eclipse is that the viral genome is sensitive to nucleases, which will gain access to the viral genome and degrade it if the cell is lysed. Thus, eclipse is defined as the stage of the viral growth life cycle where 1) viral info is present in the form of free nucleic acid and 2) is sensitive to enzymatic degradation. Maturation: process begins with the assembly of the first infectious virus in the cytoplasm. Poliovirus is synthesized and assembled entirely in the cytoplasm, matures and becomes infectious while still in the cell. The whole crop of mature virus particles stays in the host cell until it disintegrates, liberating the progeny virions. Maturation and release are separated by a period of retention that varies based on the host cell. Release: as mentioned above, progeny virus accumulates inside the host cell during maturation (this is characteristic of many naked viruses). Release is the sudden burst of the cell, producing free virus.
Additional notes: Poliovirus replication: begins with the uncoating step of the poliovirus life cycle. Following uncoating, the liberated ss (+) RNA must associate with cellular ribosomes for translation. The RNA codes for four different proteins: 1. inhibitors of cellular RNA and protein synthesis: these help kill off the host cell by interfering with cellular metabolism 2. an RNA-dependent RNA polymerase: the host cell does not have an RNA-RNA polymerase, so it must be encoded by the virus itself a. RNA-dependent RNA polymerase first synthesizes complementary (-) RNA strands b. From these copies, more (+) RNA strands are made. As multiple (+) strand RNAs are being made by RNA-RNA polymerases from a single (-) RNA template, the collective structure is known as the replicative intermediate c. Structures that contain a copy of both (+) and (-) RNA strands are known as the replicative form d. Ultimately, (+) RNA is made in large excess over (-) RNA. The (+) RNA is either incorporated into virus particles or serves as mRNA for synthesis of viral proteins. The protein product from translation is known as the polyprotein. Since the poliovirus RNA is polycistronic and codes for multiple proteins, the polyprotein must cleaved by viral proteases to yield functional proteins (see below for more info on VP0, VP1, VP3 cleavage and pentamer assembly) 3. virion structural (capsid) proteins 4. a protease needed for virus assembly and maturation
viral proteases cleave the polyprotein produced from translation to yield functional proteins In summary, translation of RNA sets in motion events that cause viral replication and cell death, which is accomplished via inhibition of cellular metabolism and the onset o viral RNA synthesis.
a.
Map of the polivorius genome: the genome consists of three domains P1 is the 5 domain and codes for capsid structural proteins P2 is located in between P1 and P3, and codes for inhibitors of cell metabolism and the small protein covalently linked to 5 end of RNA; also contains info for a second protease P3 is the 3 domain and codes for protease that participates in proteolytic processing of the polyprotein Components of the poliovirus capsid: the capsid contains four different structural proteins (VP1, VP2, VP3, and VP4). VP2 and VP4 are derived from cleavage of VP0, which takes place at the very end of poliovirus replication (see below). Capsid assembly starts with the synthesis of the 5S protomer, which contains VP0, VP3, and VP4 Five protomers combine to form a 14S pentamer, and twelve pentamers assemble into a 73S procapsid, which combines with progeny RNA to form the provirion. The provirion VP0 is cleaved to form VP2 and VP4, and its this cleavage that turns the virus into the infections 15S form. Although poliovirus maturation has elements of self-assembly, cellular participation is still needed. The cell contributes an unidentified morphopoietic factor, and the smooth ER membrane plays a role as well.

Genetic Class III and IV

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11-12-09 RNA Viruses: Genetic Class III and IV (+) stranded and dbl stranded genomes 1.

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