1. Attachment, Penetration, and Uncoating
(How Does the Virus Get In?)

2. Attachment (general)
Host Range- Refers to the different types of cells or organisms that the virus can infect.
The host range may be broad (may infect many different hosts) or very narrow (may only infect specific target tissues within a particular host)
Tropism- A virus is said to have a tropism for a particular cell type when it targets and infects that cell type.

3. Attachment (general)
The specificity of a virus for a particular host or cell type is due mainly to whether or not the target cell has the receptor to which the virus can bind to initiate an infection.
Remember that the cell must also provide the machinery that the virus requires to replicate.
If the virus successfully replicates in the host cell, the infection is productive and the host cell is said to be permissive for the virus.
If the cell has the receptor, but lacks something required for viral replication, the infection is abortive or non-productive and the host cell is considered to be non-permissive for the virus.

4. Attachment (general)
The part of the virus that binds to the host cell is called a ligand or virus attachment protein.
Ligands are usually proteins or glycoproteins.
The part of the host cell to which the virus binds is called the receptor.
The receptor may be a protein, a glycoprotein, or a lipid.
Receptors are molecules that have a role in the normal functioning of the cell, and viruses have evolved to take advantage of them.

5. Attachment (general)
Viruses may bind up to three different receptors on the cell surface in succession.
Low affinity receptors - are found in high abundance and may serve to get the virus out of the fluid bathing the cell and into intimate contact with the cell surface.
Primary receptor – are often found in in lower concentration than the low affinity receptors
Co- or secondary receptor – the virus-primary receptor interaction must occur before the virus binds to this receptor

6. Attachment (general)
The interaction between the viral ligand and the host receptor(s) during attachment or adsorption is mainly electrostatic in nature. Evidence for this is that the interaction:
May require a specific pH
May require a specific ionic strength
May require the presence of specific ions in the media.

7. Attachment (general)
There are several factors that may influence the efficiency of attachment:
Density of the receptors
Absolute concentrations of the viruses and the host cells
It is possible to synchronize a virus infection by mixing the virus with the host cells for a short period of time and then quickly diluting the virus.
All the viruses should be going through the same step in the viral replication cycle at the same time.

8. Attachment (general)
A single virus is theoretically sufficient to infect a single cell. The multiplicity of infection (moi) is equal to the number of pfu added per cell.
The efficiency of plating (EOP) is the proportion of virions that produce infected cells.
The infection of animal cells by viruses is highly inefficient, so the efficiency of plating is usually quite low.

9. Attachment (general) There are two stages in the attachment process
Reversible (binding to low affinity receptor)
Irreversible (due to alterations in the virus or on the host cell surface after binding to the primary receptor occurs)

10. Viral attachment (adsorption)

11. Penetration and Uncoating
Penetration is the mechanism used by the virus to gain entrance into the host cell.
Uncoating is, in the most simple terms, the separation of the nucleic acid from the capsid.
For some viruses there is never a total separation of the nucleic acid and the capsid because some of the capsid proteins form part of the replication complex of the virus.
For these viruses uncoating refers to changes that occur to make the viral nucleic acid ready for expression.

12. Attachment of Animal Viruses
Naked animal viruses
The best understood interactions occur between picornaviruses (small RNA viruses) and their cellular receptors.
Rhinoviruses – A deep cleft known as a canyon occurs in each triangular face of the capsid and is formed by the capsid proteins VP1, VP2, and VP3.
This serves as the ligand which binds to the receptor, ICAM –1 (intracellular adhesion molecule 1), which normally functions to bind cells to adjacent substrates.

13. Rhinovirus attachment

14. Rhinovirus attachment

15. Rhinovirus attachment

16. Attachment of naked animal viruses
Poliovirus – the ligand is a trough that runs along each corner of the virus and the receptor is CD155, a member of the immunoglobulin superfamily (all members of the family are integral membrane proteins involved in recognition and binding activities relating to the immune response or cellular communication).
Note that for the naked viruses the ligand for the virus resides on the capsid.

17. Adsorption of a Naked Virus to the Host Cell Membrane

18. Attachment of Animal Viruses
Enveloped animal viruses – for enveloped viruses the ligand is usually a glycoprotein spike that spans the envelope of the virus.
Influenza virus – The ligand on influenza virus is the hemagglutinin glycoprotein spike protein which is a trimer.
The 3-D structure has been determined by X-ray crystallography.
Each monomer is composed of 2 peptide subunits, HA1 and HA2.
The globular head (part of HA1) contains a conserved region that binds to the N-acetyl neuraminic acid (sialic acid) cellular receptor.
Sialic acid residues are found on many cell types, including RBCs.
The name hemagglutinin comes from the finding that this protein is responsible for the hemagglutination or clumping of RBCs that is caused by this virus

19. The HA Glycoprotein Spike Protein of Influenza Virus

20. Enveloped Animal Viruses
Also found on the surface of influenza virus is another spike protein, the neuraminidase protein.
This molecule is a tetramer and it functions to cleave sialic acid residues.
It is believed to function in preventing the virus from sticking to the host cell or to other viruses during exit of the virus from the host cell.
Sendai virus – Attachment occurs via the hemagglutinin-neuraminidase (HN) protein which has both the hemagglutinating and neuraminidase functions on the same protein.

21. Sendai virus attachmentHN

22. Enveloped Animal Viruses
HIV – There is recent evidence suggesting that a host cell protein incorporated into the virus during assembly, cyclophilin A, serves to initially bind the virus to the low affinity receptor heparin sulfate and this is followed by the binding of the viral ligand, gp120, to CD4.
CD4 is found on T helper cells, macrophages, and glial cells.
The binding of gp120 to CD4 results in a conformational change of gp120 which then binds to a chemokine coreceptor, CXCR4 on T lymphocytes, or CCR5 on macrophages.
CD4 is involved in the recognition of MHC antigens on B lymphocytes and functions in the immune response in which antibodies are made.
Chemokine receptors are receptors of chemical messages involved in cellular communication.
Individual HIV strains are classified as being either lymphotropic or macrophageotropic based on which chemokine receptor the virus recognizes.
Rare individuals who are resistant to HIV may have mutations in their chemokine receptors.

23. HIV binding to CD4 and chemokine receptors
gp 120
CD4

24. Adsorption of an Enveloped Virus to the Host Cell Membrane

25. Attachment of Animal Viruses
Non-specific or inappropriate interactions can accidentally result in virus being taken up into cells:
Pinocytosis – virus taken up in the fluid phase
Antibody coated virus binds via the Fc portion of the antibody to Fc receptors on the surface of monocytes and other cells – known as antibody enhancement of virus uptake.
Instead of neutralizing the virus, the antibody has inadvertently increased its pathogenicity.

26. Penetration and Uncoating of Animal Viruses
Penetration and Uncoating may be coupled events or they may be separate events.
Penetration is an energy dependent process and requires that the host cell be metabolically active. There are three main mechanisms of penetration by naked viruses:

27. Mechanisms of Penetration
Naked viruses:
Translocation of the entire virus across the cytoplasmic membrane which is mediated by capsid proteins and specific membrane receptors.
This mechanism is rare and poorly understood.

28. Translocation

29. Mechanisms of Penetration
Endocytosis into intracellular vacuoles –This is the most common method of entry. It relies on the normal cellular mechanism of receptor mediated endocytosis.
The virus binds to its receptor which may already be present in clathrin coated pits.
If the receptor is not already present in a clathrin coated pit, the binding of the virus triggers the movement of the receptor to a clathrin coated pit.
There is an increasing invagination of the pit and an eventual pinching off of the membrane to form an intracellular endosome containing the virus (penetration)

30. Endocytosis
The endosome (coated vesicle) becomes acidified and clathrin is released from the vesicle.
The lowered pH in the vesicle causes conformational changes in the capsid proteins leading to release of a hydrophobic region that interacts with the vesicle membrane forming a pore.
The nucleic acid alone or the nucleic acid plus associated proteins move thru the pore into the cytoplasm.

31. Endocytosis

32. Endocytosis

33. Endocytosis
For adenovirus, viral capsid proteins are sequentially removed in the acidified endosome, but some capsid protein remains associated with the genomic DNA as it is released into the cytoplasm where it attaches to microtubules.
The capsid protein-DNA complex moves along the microtubules to the nucleus and the genomic DNA enters the nucleus through a pore (uncoating).

34. Adenovirus penetration and uncoating

35. Mechanisms of Penetration
Forming a pore in the host cell plasma membrane
The attachment of poliovirus to its receptor leads to major conformational rearrangements in the viral particle.
VP4 in the poliovirus is released to interact with the cell membrane.
A previously hidden hydrophobic domain of VP1 also interacts with the membrane to form pores through which the viral genome is released into the cytoplasm (uncoating).
It is unclear whether the release of the poliovirus RNA occurs at the plasma membrane or from within endosomes. A decreased pH is not required.

36. Poliovirus penetration and uncoating

37. Polio virus penetration and uncoating

38. Penetration by rearrangement of capsid proteins

39. Mechanisms of Penetration
Enveloped viruses – rely on a viral fusion protein which contains a fusion peptide that inserts into a cellular membrane to mediate fusion of the membrane with the viral envelope during the fusion process. Most fusion proteins:
Are integral membrane glycoproteins
Are synthesized as a precursor that must be post-translationally cleaved to participate in fusion.The tropism of the virus is often determined by whether or not the host or tissue contains an enzyme capable of this cleavage.
Contain a highly conserved fusion peptide that is released upon cleavage of the fusion protein.
The fusion peptide is found on the subunit of the protein containing the transmembrane domain.

40. Enveloped viruses
Fusion of the viral envelope can occur at the cell surface (pH independent fusion) or after endocytosis (pH dependent fusion)
pH independent fusion
Sendai virus- fusion of the viral envelope with the plasma membrane is mediated by the fusion protein and the nucleocapsid in released into the cytoplasm for replication.
The capsid proteins are involved in the replication process, so there is never a total separation of the capsid from the genomic RNA.
Penetration and uncoating appear to be coupled.

41. Sendai virus fusion at the cell membrane

42. Fusion at the cell membrane (pH independent)

43. Fusion at the cell membrane (pH independent)
Herpes simplex viruses I and II – fusion is mediated by gpB and gpD.
The nucleocapsid is released into the cytoplasm (penetration) and is transported to the nucleus via its interaction with cytoskeletal elements.
DNA is released through pores into the nucleus (uncoating)
HIV ( a retrovirus)- fusion is mediated by gp41 which is formed when gp160 is cleaved to gp120 and gp41 (remember that gp120 is the ligand for attachment).
CD26 on the host cell surface appears to play a role in the fusion process.
Following fusion, the nucleocapsid is released into the cytoplasm (penetration)

44. Retroviral penetration
Retro-virus
HIV

45. HIV penetration

46. Fusion at the cell membrane (pH independent)
For HIV, cyclophilin A, a host cell protein that facilitates protein folding and assembly (a chaperone) and possesses peptidylprolyl isomerase activity, plays a role in viral uncoating. (It should be noted that there is also evidence that it plays a role in low affinity attachment of the virus to heparin sulphate)
Cyclophilin A is incorporated into newly formed virions.
Virions lacking cyclophilin A are not infectious because they are blocked in early replication before reverse transcription takes place.
It is postulated that the viral core must expand before reverse transcription can take place and that cyclophilin A may play a role in the process of expansion by facilitating the release of some viral protein.

47. Model for HIV uncoating

48. pH dependent fusion pH dependent fusion (fusion after endocytosis)
Orthomyxoviruses (Influenza) enter the host via receptor mediated endocytosis.
When the pH in the endosome decreases, there is a conformational change in the HA of influenza which exposes the fusion peptide that was previously hidden within the trimer.
The peptide mediates fusion of the viral envelope with the endosomal envelope, thus releasing the nucleocapsid into the cytoplasm (penetration).
The nucleocapsid is transported into the nucleus via a nuclear localization signal on the nucleoprotein (uncoating).

49. pH dependent fusion

50. Influenza virus penetration and uncoating

51. Conformational change in HA at low pH

52. pH dependent fusion