Lecture 8 - Negative Strand RNA Viruses

Lecture 8 - Negative Strand RNA Viruses

• Filoviruses are characterized by their filamentous, enveloped structure.
• Filovirus diameter is 80 nm, and length can exceed 800 nm.
• Filoviruses have a negative-sense single-stranded RNA genome, approximately 15-19 kb in size.
• Their genome encodes proteins such as nucleocapsid protein (NP), glycoprotein (GP), RNA polymerase cofactor (VP35), polymerase (L), matrix protein (VP40), and VP24

Filoviruses - Structure

• Filoviridae derives from the Latin word "filum" (filament or thread-like).
• These viruses are filamentous and enveloped.

Filoviruses - Disease and Spread

• The first Marburg virus outbreak occurred in Marburg and Frankfurt, Germany in 1967.
• Laboratory workers processing monkey tissues were affected, with a 22% mortality rate (7 deaths out of 32 cases).
• A similar virus, Ebolavirus, emerged independently in Zaire (now the Democratic Republic of Congo) and Sudan in 1976.
• Different Ebolavirus species exist, including Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV).
• Marburg and Ebola viruses are sporadically re-emerging viruses causing severe, often fatal disease in Africa.
• A 1994 outbreak originated from Cote d'Ivoire, resulting in the third EBOV species (CIEBOV or Tai Forest).
• Experimental monkeys shipped from the Philippines to Reston, Virginia (USA) in 1990 harbored a non-pathogenic ebolavirus (REBOV).
• Bundibugyo Ebola virus (BEBOV) was discovered in Uganda in 2007.
• The 2014-15 EBOV outbreak was the largest in history, originating in Guinea.

Filoviruses - Genome and Proteins

• Filovirus genomes are linear, negative-sense, single-stranded RNA (~15-19 kb).
• Seven genes transcribed in series from 3' end of the genome using viral RNA polymerase.
• Most genes produce a single mRNA and a single protein (except glycoprotein GP, which is cleaved into multiple proteins).
• Proteins of filoviruses:
  • Nucleocapsid protein (NP)
  • RNA polymerase cofactor (VP35)
  • Matrix protein (VP40)
  • Glycoprotein (GP, cleaved into GP1, GP2, or sGP)
  • Membrane protein (VP24)
  • RNA polymerase (L)
• Glycoproteins are either secreted glycoprotein (sGP) (making up 80%) or GP (20%).

Filoviruses - Replication

• Transcription and replication occurs in the cytoplasm.
• The template for mRNA synthesis is the negative-sense RNA genome.
• The 3' leader contains a promoter for viral RNA polymerase and packaging signal for nucleocapsid assembly.
• During replication, viral RNA polymerase adds a methylated 5' cap and poly(A) tail.
• Genome replication occurs through the synthesis of antigenome RNA.
• Viral RNA polymerase changes its mode to no longer recognize termination/polyA-re-initiation sites.
• Genome RNA is packaged into nucleocapsids during replication.

Filoviruses - RNA Editing

• Ebolavirus uses RNA editing to produce two glycoproteins (sGP and GP) from a single gene.
• sGP (~80%) is a shorter protein without the transmembrane domain located at the C-terminus.
• GP (~20%) has an extra adenine (A) residue.

Filoviruses - Attachment and Entry

• The glycoprotein (GP) is synthesized as a precursor protein and inserted into the endoplasmic reticulum (ER).
• An N-terminal signal sequence is cleaved off during the insertion in ER.
• GP undergoes extensive glycosylation in ER and Golgi and transported to the plasma membrane.
• Cellular furin protease cleaves GP into ectodomain, GP1 (N-terminal) and transmembrane GP2 (C-terminal) proteins.
• GP1 and GP2 are held together by a disulfide bridge (between cysteine residues).
• Filovirus GP mediates binding to multiple cellular receptors.
• Cellular receptors include, asialoglycoprotein receptor, folate receptor–a, integrins, and DC-SIGN.
• Filovirus pseudotypes can be used to study attachment and entry.

Filoviruses - Protein Function

• sGP is released from infected cells and found in serum, acting as a biomarker and potential vaccine/antiviral target.
• sGP's function is unclear; it may substitute as a structural protein or influence replication and infectivity.
• VP40 is the most abundant viral protein and is associated with the viral envelope (located at the cytoplasmic side of the plasma membrane or inner side of the viral envelope).
• VP40, similar to other enveloped virus matrix proteins, bridges envelope glycoproteins to nucleocapsids.
• Expressed VP40 in mammalian cells leads to virus-like particles budding from the plasma membrane.
• Cellular proteins involved in trafficking/sorting of intracellular vesicles (e.g., Nedd4, Tsg101) interact with VP40 in the formation of virions at the plasma membrane.

Influenza Viruses - Structure

• Orthomyxoviridae is derived from Greek 'ortho' (correct/normal) and myxa (mucus).
• The virus is characterized by its enveloped particles, quasi-spherical to filamentous, in shape derived from host membrane via budding.
• Viruses have a diameter of 80-120 nm, and compact helical nucleocapsids.

Influenza Viruses - Types

• Subtypes are distinguished by variations in surface glycoproteins (hemagglutinin (HA) and neuraminidase (NA)).
• Avian influenza viruses (e.g., H5N1, H7N9) can be highly pathogenic, whereas low pathogenic avian influenza viruses have decreased ability to kill chickens in labs.
• Influenza A viruses, most commonly, infect humans. Influenza B viruses mostly infect humans.
• Other genera found in Orthomyxoviridae family, include Thogotovirus and Isavirus.

Influenza Viruses - Disease and Spread

• Influenza A virus infections typically lead to symptoms such as high fever, sore throat, cough headache, and muscular pain, often with secondary bacterial infections in the elderly, infants, and those with chronic illnesses.
• Influenza A viruses cause sporadic pandemics and annual epidemics.
• Avian influenzas are a continual threat to birds, and have potential to infect humans, leading to outbreaks.

Influenza Viruses - Genome

• Influenza viruses have segmented, negative-sense RNA genomes (6-8 different segments).
• Single virions house a complete set of genome fragments.
• The genome size is approximately 10-15 kb.

Influenza Viruses - Genome and Proteins

• Influenza A virus genome has multiple RNA segments (with corresponding proteins).
• Proteins include polymerase complex (PB2, PB1, PA), nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 ion channels, nuclear export protein (NEP).

Summary

• Virus classifications are used to understand their characteristics, evolution, impact and behaviour.