Lecture 8

Question 1

filovirus structure

Answer 1

• Filamentous, enveloped particles
• fairly large viruses but have small diameter
• Helical nucleocapsids with negative sense ssRNA genome
• have large genome

Question 2

filovirus genome

Answer 2

• Linear, negative sense, single-stranded RNA genome (‘-’ ssRNA)
• Seven genes, transcribed in series from 3’ end of genome by viral RNA polymerase
• seven genes in a conserved order
• Most genes produce a single mRNA and a single protein, except for GP → gets cleaved into GP1 and GP2 or sGP
  (secreted GP)
• All genes flanked by conserved sequences that signal transcription termination, polyadenylation and reinitiation
• Individual transcripts made for each viral protein, RNA synthesized for each protein

Question 3

disease and spread of filoviruses

Answer 3

• Two genera: Marburgvirus and Ebolavirus
• spread among humans is limited to close contacts
• Aerosolized human-human spread is low risk
  Person-person transmission is mediated by physical contact with secretions/excretions
  e.g. blood, feces, vomit, urine, semen
• Close family members and medical staff are the most at-risk
• Epidemics usually are self-limiting, as virus transmission period is transient (slow transmission) and generally ineffective

Question 4

proteins packaged in the virions

Answer 4

• Nucleocapsid protein (NP)
• RNA polymerase cofactor (VP35)
• Matrix protein (VP 40)
• Envelope glycoproteins (GP, cleaved into
  GP1, GP2 or sGP)
• Minor nucleocapsid protein (VP30)
• Membrane protein (VP24)
• RNA polymerase (L)
• Ebola makes additional secreted glycoproteins
  (sGP, delta-peptide)

Question 5

how are filovirus proteins named?

Answer 5

based on function, glycosylation status or molecular weight

Question 6

what proteins are associated with genomic RNA/ and or nucleocapsid? (ebola virus)

Answer 6

• NP (nucleoprotein)
• VP35
• VP30
• L (large protein)

Question 7

what proteins are associated with the viral envelope? (ebola virus)

Answer 7

• VP40
• GP
• sGP
• VP24

Question 8

filovirus replication

Answer 8

• Transcription and replication occurs in cytoplasm
• template for mRNA synthesis is the ‘-’ sense RNA genome
• 3’ leader contains promoter for viral RNA polymerase and packaging signal for assembly of nucleocapsids
• continuous system

Question 9

what determines whether transcription or replication occurs?

Answer 9

• level of NP protein
• transcription occurs at low levels
• replication occurs at high levels

Question 10

filovirus transcription steps

Answer 10

• stop & go system
  1. Viral RNA polymerase begins
  transcription at 3’ terminal of
  the genome
  2. little or no free NP
  protein (during initial infection)
  is present → RNA polymerase
  transcribes a short sequence,
  then terminates to release a free
  leader RNA and then scans for
  nearby mRNA start site and reinitiates transcription at the next
  gene (re-initiation is not 100%
  efficient)
  3. L protein adds methylated 5’ cap
  and polyA tail, like cellular
  mRNAs
  4. Once sufficient NP made,
  genome replication can begin

Question 11

inclusion bodies

Answer 11

• in cytoplasm of infected cells
• contain viral nucleocapsids
• present sites of viral replication, protein aggregation, or the assembly of viral particles
• In the case of filoviruses, such as Ebola virus and Marburg virus, inclusion bodies play a critical role in the virus life cycle

Question 12

filovirus RNA editing

Answer 12

• make two glycoproteins from the same gene
• sGP (~80%) and GP (20%)
• editing site contains a stretch of 7 ‘U’s → transcribed into ‘A’s in ‘+’ sense mRNA
• viral RNA polymerase will ‘stutter’ over this stretch of ‘U’s and add an additional ‘A’
• changes Thr to Asn and changes the reading frame downstream.
• some cases, it may be 6 or 9 ‘A’s producing a smaller sGP (ssGP)
• Editing of Ebolavirus gene product can produce sGP (most common), GP, ssGP (least common

Question 13

sGP

Answer 13

• 80%
• shorter protein that does not include the transmembrane domain at the C
  terminus
• is secreted, cannot stay in the membrane
• yield delta-peptide upon proteolytic cleavage

Question 14

GP

Answer 14

• 20%
• RNA polymerase “stutters” and adds an extra ‘A’ residue to change the reading frame to yield full length GP

Question 15

filovirus attachment

Answer 15

• Filovirus GP mediates attachment and entry (by fusion)
• GP mediates binding to multiple cellular receptors

Question 16

how GP gets to the plasma membrane

Answer 16

• synthesized as a precursor protein, inserted into the lumen of ER:
• N-terminal signal sequence is cleaved off during insertion in ER
  Protein undergoes extensive glycosylation in ER and Golgi
  → transport to plasma membrane
• Cellular furin protease then cleaves GP into the ectodomain GP1 (N-terminal) and transmembrane GP2 (C-terminal)
• GP1 and GP2 held together by a disulfide bridge between two cysteine residues (cysteine bridge)
• GP forms trimers at plasma membrane and in the envelope (virion surface)

Question 17

what cellular receptors can GP mediate binding to?

Answer 17

Asialoglycoprotein receptor: Liver-specific, binds to and internalizes glycoproteins that lack terminal
sialic acid
* Folate receptor-α: binds to folic acid
* Integrins: cell-surface proteins that interact with extracellular adhesion proteins and initiate intracellular
signalling
* DC-SIGN: dentritic (immune cells) cell-specific intracellular adhesion molecule-grabbing nonintegrin – type II
transmembrane proteins that bind mannose and involved in interaction with T cells

Question 18

pseudotypes

Answer 18

• “artificial virus”
• do not have capacity to replicate
• recombinant viral particles that combine the structural components of one virus with the envelope proteins of another

Question 19

how can attachment and entry be studied?

Answer 19

• Filovirus pseudotypes have GP incorporated into the envelopes of unrelated recombinant viruses
• can be used to study viral proteins

Question 20

why is the pseudotype technique problematic to study attachment and entry?

Answer 20

• not an actual functional Ebola virus being used
• adding a GP to an unrelated virus would require you to control for processes created by the unrelated virus

Question 21

entry of filoviruses

Answer 21

• taken up via macropinocytosis
• using its fusion peptide (N-terminal of GP2) it fuses within vesicles to enter cells (probably a low pH trigger
• virus binds to the receptor which triggers signalling cascades within the cell
• this changes the polymerization of actin causing it to form a vesicle around the virus for it to be internalized

Question 22

function of sGP

Answer 22

• released from infected cells and is found in serum of infected patients so it can be used as a biomarker and as a potential vaccine / antiviral target
• Function is not entirely clear
• Considered non-structural but may substitute as a structural protein by forming a complex with GP2
• may limit GP cytotoxicity → more efficient replication and infectivity
• Acts as a soluble factor that targets elements of the host defence system e.g. binding to antibodies and contribute to immunosuppression

Question 23

VP30 function

Answer 23

• Minor nucleocapsid protein
• activates viral mRNA synthesis in Ebola virus
• mechanisms of action is unclear
• stem-loop structure at the beginning of the NP gene inhibits RNA polymerase from initiating mRNA synthesis → VP30 reverses this inhibition

Question 24

VP40 function

Answer 24

• most abundant viral protein and is associated with viral envelope
• Located at cytoplasmic side of plasma membrane and/or inner
  side of viral envelope
• bridges envelope gp’s to nucleocapsids
• sufficient to form viral envelope structures and virus particles
• Cellular proteins that are involved with trafficking and sorting of
  intracellular vesicles may interact with VP40 to form virions at plasma membrane

Question 25

recrudescence

Answer 25

recurrence of disease / symptoms
after a period of inactivity