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PicornavirusComments

Picornavirus

The picornavirus family has these general properties:

  • Large groups of viruses with common structure, genome organization, and expression
  • Genome is a single strand of + sense RNA enclosed in an icosohedral non-enveloped capsid
  • Causes a wide range of diseases including poliomyelities, enteitis, hepatitis, and common colds.

The picornavirus family has these 5 genuses, with a popular species of that genus listed next to it:

  • Enterovirus (poliovirus)
  • Rhinovirus (human rhinovirus 1A)
  • Hepatovirus (hepatitis A virus)
  • Cardiovirus (encephalomyocarditis virus)
  • Aphtovirus (foot and mouth disease virus)

Poliovirus will be examined more closely as a model organism for the picornavirus family. As the first virus to be grown in culture (by Dr. Enders in 1949), it has been heavily studied. There are 2 vaccines used to combat poliovirus:

Salk's inactivated virus
  • The virus is inactivated by heat and formaldehyde treatment.
  • It is less efficient, granting only humoral immunity (immunity via blood and lymphatic fluid).
  • It is short-lived, but low-risk.
Sabin's attenuated virus
  • It is a live non-pathogenic mutant.
  • It is long-lasting, oral, and grants both humoral and cellular immunity.
  • Receipients shed attenuated virus, which can mutate to become pathogenic. Hence, it is risky.

The course of infection is as follows:

  • Like all enteric viruses, it is secreted in feces.
  • Fecally contaminated food and water transmit the virus.
  • Once the virus is ingested, it infects the gut.
  • The primary viremia (first time the virus enters the bloodstream) leads to fever, drowsiness, headache, nausea, and vomiting, followed by recovery within a few days.
  • However, in rare cases, poliovirus can invade the CNS and target neurons. Paralysis ensues due to neuronal damage.

There are many ways for the virus to be transmitted. Although usually it results from sewage leaking into a water supply, it can also happen if somebody does not wipe well and then goes swimming in a pool. Since poliovirus can be such a debilitating disease, vaccination is very important. However, it is not necessary to vaccinate everybody. By a phenomenon called herd immunity, where just enough people are treated to block transmission, the disease can be eradicated. Continued vaccination is crucial, though, because lab stocks are maintained (and can be released) and vaccinated patients shed attenuated virus (which can mutate and become pathogenic).

In the absence of any viral proteins, the poliovirus nucleic acid can still produce all proteins needed to make new virion particles. If it had been minus sense, it would need to be transcribed before use as a template for translation.

The 5' end of the genome is highly structured. Poliovirus contains a very small protein at the 5' end called VPg. VPg is attached covalently (phosphodiester bond) to the uridine residues at 5' end of the RNA chain. VPg is present in packaged virion RNA but not on viral RNA that is being translated by cellular ribosomes. Upon infection, a cellular enzyme cleaves VPg from VPg-containing viral RNA. poly(A) is also unusual because it is encoded in the genome, as opposed to being added after transcription. This means it is genetically coded (copied from poly(U) at 5' end of minus strand during replication) and not added post-transcriptionally.

The CAP structure is crucial for positioning mRNA on ribosome for translation. How does the poliovirus RNA translate? Instead of the ribosome binding to the 5' CAP, the ribosome binds internally to the 5' UTR. It is highly structured, with many hairpin loops. There is the VPG, several loops, then the Internal Ribosome Entry Site (IRES) or Ribosome Landing Pad (RLP). At the 3' end of the IRES is the initiating AUG. There is only one ORF in polio RNA, and no stop codon between protein coding sequences. This indicates the presence of a single long polyprotein. Internal binding selects 9th AUG in poliovirus RNA (same as mRNA). Mature viral and structural proteins are generated by proteolytic cleavage of a long precursor polypeptide (polyprotein). Cleavages occur fast, so it is difficult to detect this large polyprotein. Strucutural proteins on left translated before non-structural proteins on right. Ineffcient. Non-structural proteins like RNA-dependent RNAP 3Dpol not needed in high oconcetrations.

  • Viral 2A proteasse functions while still part of polyprotein precursor. Before termination, it auto-catalyzes cleavage of P1 from the growing polypeptide chain.
  • Viral 3C protease is on right end of genome and catalyzes generation of individual proteins
  • Viral proteases also function to shut down host cell translation and transcription, thus allocating more resources to viral processes.
    • Eukaryotic translation: unwinding beings at 5' end by binding of the 5' cap by cellular protein complex (cap-binding protein, CPB, or eIF-4F) which consists of eIF-4A, 4B, and 4E-p220). Poliovirus lacks a CAP, so it doesn't depend on p220 for it's function. 5' UTR secondary structure recognized by cellular p52 and other proteins. Still recruits eIF4a and 4B to unwind secondary structure. Ribosome binds internally. So, cleavage of p220 by viral 2A inhibits cellular protein synthesis but not poliovirus protein synthesis.
    • eIF2 is involved in recruiting tRNAmet-Met to ribosome and positioning the initiator tRNAmet onto AUG. In this energy-dependent process, Gtp bound to eIF2 is hydrolyzed to GDP. Recycling of eIF2 requires exhange of GFP for GTP to start antoher round of recruitment. GEF (GDP exchange factor) catalyzes this. eIF2 recruits met-tRNA to the 40s ribosome and poliovirus proteins block eIF2 recycling. eIF2 phosphorylation prevents guanine nucleotide exhachange, nblocking recruitment of tRNA to reibosome and formotation of 40s complex.
    • Also, trancription is halted. Viral protease 3C is translocated and cleaves transcription factor TBP (TATA-binding protein) and other factors, leading to cellular transcription shut-off.
    • Poliovirus protease 2A shuts off translation by phosphorylated eIF2 and cleaving p220; poliovirus protease 3C shuts off transcription by TBP and TFIIIC.

The fate of newly synthesized plus sense RNA depends on the time after infection. Newly synthesized plus sense RNA has 3 fates:

  • Translation (early)
  • Replication Template (requires viral replicase)
  • New Viral Genomes (late)

The viral replicase, an RNAP, is unable to initiate synthesis from RNA. This is unusual. Like DNAP, it needs a primer. VPg-uridine (VPg attached to uridine) serves as this primer. The viral replicase copies +RNA to make complementary -RNA. This minus strand is the template for many new plus strands. The replicative intermediate (RI) is one minus strand template hydrogen bonded to many nascent plus strand RNAs and vice-versa.

There are 5 miscellaneous topics which are nonetheless very important:

Capsid StructureThere is a canyon....at this canyon is where neutralziing antibodies bind. MPressure is for virus to matuate resuidues to avoid immune surveillance. Both polio and rhinovirus have canyon. This repesents hyighly conserved sequences suggesting essential function, including binding to cellular receptor. VIrus retains such functionally important residues, but buries them too deep for natibodies to bind thre. WIN compounds treat rhinovirus infections. Polioviral capsid proteins have a commons structure of eight stranded antiparallel β barrel.
Receptor StructureICAM-1 inter cellular adhesion molecules. Five extracelllular IgG-like domains with disulfide bonds at N-terminuis, membrane spanning domain, and cytoplasmic ce-terminal domain. PIliovirus recetpor looks same expect 3 extraceullar IgG-like domains.
Poliovirus Receptor Isolation ExperimentPurify protein from cell surface intereacting with virus in solution. Protein is receptor confirmed by avility of anti-receptor antibody to block infection of receptor-positive human cells by poliovirus.
Mouse L cell ExperimentMouse L cells do not express poliovirus receptor and not susceptible to infection. Transfect with human cDNA library and human chromomosomes contain PVR and mouse cells trasnfected with human genes expresssed PVR on cell surface allowing poliovirus to bind and infect human cells.
Baltimore's Cleavage-Blocking ExperimentUsually there is the polio + strand RNA which is converted to NH2-------polyprotien (NCVP)----COOH which by proteolytic cleavage becomes P1, P2, P3. P1 has VP0 (VP4 and VP2). Canavine fluorphenylalanine (CFP) blocks cleavage of the polyprotein into P1, P2, and P3. Baltimore showed the existence of a polyprotein by infecting cells and incubating in CFP. Proteins were isolated, and labelling indicated presence of one long polyprotein.