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Viral Pathogenesis > Viral Attachment and Entry > Flashcards

Viral Attachment and Entry Flashcards

1
Q

One common goal of viruses

A

To reproduce. Viruses are obligate intracellular parasites, so they rely on a host cell to be able to do this.

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2
Q

Steps in viral entry (7)

A
  1. Viruses randomly collide with multiple different cell types and undergo electrostatic, nonspecific reactions with them. Their surface proteins act as a “pass” to get into the target cell
  2. Viral proteins allow them to bind to the host cell surface receptors, triggering internalization
  3. The virus is internalized
  4. Intracellular processing
  5. Vesicular trafficking- the virus can move along microtubular structures to the site of replication (cytoplasm or nucleus)
  6. Nuclear transport
  7. Viral replication
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3
Q

Viral fusion

A

Enveloped viruses can fuse with the host cell membrane, or they can be internalized and fuse somewhere else in the cell. Fusion releases the genome into the cell or the subcellular compartment

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4
Q

Cellular receptors for viral attachment

A

These receptors are essential for all viruses, except for viruses that infect fungi and plants. They are unique to get virus. Adenovirus has fibers projecting from penton units. Influenza A has hemagglutinin proteins that recognize cell surface receptors. In HIV, gp120 recognizes host cell receptor CD4

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5
Q

Attachment factors

A

Sugar modifications- these include heparan sulfate proteoglycan (HSV) and sialic acid (influenza). They act as receptors for their respective viruses

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6
Q

Examples of viral receptors (5)

A

Includes cell surface protein receptors
1. GM1 ganglioside (SV40)
2. Ldir (Rhinovirus)
3. DC-SIGN (herpesvirus)
4. Integrin alpha-beta 3 (hand foot and mouth disease)
5. CD4, CCR5, and CXCR4 (HIV)
6. Car and integrins (Adenovirus)

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7
Q

Purpose of cell receptor proteins

A

Viruses have evolved to use proteins that will not be lose from the cell. Therefore, their receptors are proteins that have important functions for the cell. The proteins do not exist solely to be viral receptors

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8
Q

Tropism

A

Viruses only target cells that have their specific receptors

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9
Q

Variety of receptors and coreceptors

A

Sometimes, a virus needs to recognize multiple receptors rather than just one. In these cases, the virus is more stringent in its specificity. While Human rhinovirus 2 only activates one receptor (LDL receptor), HIV requires multiple. It binds to CD4 first, but then must bind to either CCR5 or CXCR4 as a coreceptor. Only at this point can the steps be triggered to internalize the virus. Hepatitis C recognizes 3 receptors- CD81, claudin-1, and occludin. Two viruses may also recognize the same receptor- CAR is recognized by both Coxsackie and Adenovirus

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10
Q

Receptors vs coreceptors

A

When multiple receptors are required for productive entry, the first receptor contacted is referred to as the receptor, and the subsequent contacts are referred to as coreceptors

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11
Q

Function of coreceptors

A

The recognition of multiple receptors induces a step-wise conformational change in the virus spike glycoproteins and the plasma membrane. It also plays a role in overcoming anatomic and topological barriers

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12
Q

Do viruses of the same family always bind to the same receptors?

A

Viruses of the same family may bind to different receptors. For example, Rhinoviruses bind to 3 different receptors and Retroviruses bind to 16 different receptors

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13
Q

Do multiple receptors increase binding avidity?

A

Affinity may remain the same, but the overall binding avidity increases. This allows for stronger binding of the virus and the cell, which is required for internalization of the virus.

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14
Q

Which forces bind a virus attachment site to a receptor?

A

These interactions are non-covalent. As internalization occurs, the virus must be able to reverse its binding from the cell surface receptors. Therefore, these reversible interactions include hydrogen bonds, ionic interactions, and van der Waals interactions

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15
Q

Technologies for receptor identification (5)

A
  1. Monoclonal antibodies
  2. Recombinant DNA technologies
  3. CRISPR/Cas 9 knockout of receptors
  4. siRNA
  5. Flow cytometry- can measure the extent of infection inside of a cell
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16
Q

Monoclonal antibodies

A

Antibodies can bind to viral receptors to block infection. This can result in either no infection at all or a minimized extent of infection. You can also use antibodies to bind to all cell proteins on a given cell type if you don’t know much about the virus. Monoclonal antibodies can also be used against viral surface proteins to block them from binding to a cell

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17
Q

Natural ligand approach

A

If you know a natural ligand for a given receptor, you can use the ligand instead of a monoclonal antibody. The ligand binds to the receptor so the virus can’t bind it and cause infection

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18
Q

Recombinant technology

A

Can be used to clone a critical receptor. If a gene is delivered to a target cell using a vector, the cell will then express a certain receptor and the virus can then infect it. This experiment is used to learn which receptors are necessary for a certain viral infection. Sometimes, multiple receptors are needed for infection

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19
Q

Gene knockout

A

Crispr technology is used. If the gene is not functioning, the receptor is not expressed, and the cell will not be susceptible to infection. This experiment confirms whether a viral receptor is necessary for infection

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20
Q

Experimental evidence that ACE serves as the receptor for COVID

A

The researchers used HeLa cells and found that HeLa cells do not express the ACE2 receptor and were not susceptible to Covid infection. They then used recombinant DNA technology to make HeLa cells express ACE2 from a variety of species- human, bat, swine, cat, and mouse. These cells then stained for the presence of nucleoprotein, which indicates productive infection. However, in mouse cells, CoV2 didn’t bind to ACE2, which suggests that SARS doesn’t recognize mouse ACE2 receptor

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21
Q

How can we engineer a mouse model for nCoV-2?

A

Using transgenic mice is necessary, so they will express human ACE2 instead of mouse ACE2

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22
Q

How does poliovirus attach to receptors?

A

Polio is a non-enveloped virus. Its capsid contains indentations/grooves called “canyons”. Cell surface receptors (in this case, CD155) bind in these canyons. Experimentally, the involvement of this receptor was confirmed using recombinant DNA technology to produce soluble CD155. CD155 has 3 domains, with the top domain being able to fit into the canyons

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23
Q

How does adenovirus attach to receptors?

A

Adenovirus is a non-enveloped virus. It has 12 fibrous projections coming out of the capsid, at the penton base. The penton base has an exposed RGD (arg-gly-asp) motif that associates with integrins (the co-receptor). Trimeric projections contain a knob on the top with an affinity for the N-terminal domain of cellular coxsackie and adenovirus receptor (CAR). The penton base (not the fiber) can then bind the integrin molecules

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24
Q

How does the influenza A virus attach to receptors?

A

Influenza A is an enveloped virus that has 8 RNA segments as its viral genome. On the surface it has 2 types of glycoproteins- hemagglutinin and neuraminidase. These glycoproteins make contact with host cell surface proteins containing sialic acid residues (a terminal sugar). Hemagglutinin has a globular head that binds to sialic acid. Attachment eventually leads to entry of the virus particle

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25
Q

Sialic acid linkage and specificity for influenza

A

While sialic acid is the terminal sugar, galactose is the second to last sugar on the host cell glycoproteins. How the sialic acid is linked to galactose determines the specificity of the cell for human influenza or avian influenza. If sialic acid is linked to galactose through an alpha 2-3 linkage (carbon 2 on sialic acid is linked to carbon 3 on galactose), the receptor is specific for avian influenza- birds have this linkage in their upper respiratory tract, but humans do not. The alpha 2,6 linkage (carbon 6 on galactose) makes humans susceptible to human influenza

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26
Q

Neuraminidase function

A

Allows the assembled influenza A virus to be released from the infected cell. It can break the alpha 2-3 or 2-6 linkages between sialic acid and galactose so the virus can be released. This is important for the next cycle of viral infection

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27
Q

Tamiflu

A

An antiviral given to people with severe flu infection. It inhibits neuraminidase so the virus can’t exit the cell and infection is halted

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28
Q

How does HIV-1 attach to receptors?

A

HIV-1 is an enveloped virus with trimeric globular surface glycoprotein gp120. gp120 has a transmembrane domain called gp4. gp120 interacts with cell surface protein CD4. CD4 has a specific amino acid residue called phenylalanine 43 (Phe43) that is necessary for gp120 binding. Phe43 mutation can abolish viral interaction with the host cell

29
Q

2 receptors for HIV

A

HIV binds to receptors in a sequential manner. After binding CD4, HIV uses co-receptors CCR5 or CXCR4 (the virus only binds to one of these), present on CD4 T cells. Different variants of the virus are specific to use one co-receptor over the other. Macrophages only express CCR5, so HIV can only bind to that co-receptor to infect a macrophage. The gp120- CD4 interaction causes conformational changes that allow the virus to bind to a co-receptor and be internalized into the cell

30
Q

Mechanisms of uptake of macromolecules into cells (3)

A
  1. Phagocytosis
  2. Pinocytosis
  3. Receptor-mediated endocytosis
    Viruses can utilize all of these mechanisms, especially RME
31
Q

Virus entry into cells

A

The inside of the cell is tightly packed, making it very crowded. Therefore, the movement of large proteins or molecules can’t occur by diffusion. Transporting the virus to the site of replication can be very challenging

32
Q

Receptor mediated endocytosis

A

A form of endocytosis in which receptor proteins on the cell surface are used to capture a specific target molecule. The receptors, which are transmembrane proteins, cluster in regions of the plasma membrane known as coated pits (like clathrin). Mediated by clathrin or caveolin proteins

33
Q

Phagocytosis

A

A form of endocytosis in which large particles, such as cells or cellular debris, are transported into the cell. Giant viruses can be taken up by phagocytosis

34
Q

Endocytosis

A

Not necessary for enveloped viruses, which can fuse with the plasma membrane

35
Q

Uncoating

A

Occurs after viral attachment to a target cell. Uncoating is the release of viral nucleic acid from its protective capsid or lipid envelope (the nucleic acid is still complexed with viral proteins).

36
Q

Simple uncoating

A

Fusion of the viral membrane with the plasma membrane

37
Q

Complex uncoating

A

Passage through the endocytic pathway and docking at the nuclear membrane.

38
Q

Early fusion at the plasma membrane

A

Receptor binding, then membrane fusion- this process occurs at a neutral pH. Sequential engagement of
receptors triggers the fusion and uncoating. Thus, fusion is regulated by receptor engagement and cellular protease that cleaves the fusion
protein

39
Q

Fusion of the measles virus

A

Measles virus (MV) enters cells by membrane fusion at the cell surface at neutral pH. Two glycoproteins mediate this process: the hemagglutinin (H) and fusion (F) proteins. The H-protein binds to receptors, while the F-protein mediates fusion of the viral and cellular membranes. The H protein’s binding to receptors causes conformational changes in the F protein. The F protein then fuses the viral and cellular membranes, releasing the virus’s RNA genome into the cytoplasm.

40
Q

Fusion of HIV (3 steps)

A
  1. Viral Env glycoproteins gp120 and gp41 are trimeric spikes on the viral surface
  2. gp120 binds to CD4, causing a conformational change that forms the co-receptor binding site
  3. gp120 binds CXCR4 or CCR5 co-receptor, causing a conformational change that leads to insertion and gp41 fusion peptide into the cell membrane
41
Q

Endocytosis and fusion of influenza A (6)

A

Influenza A is taken up by endocytosis. HA viral protein interacts with sialic acid on the host cell membrane
1. The virus is internalized in an endosome, which travels in the interior of the cell, along microtubules, using kinesis proteins
2. The endosome becomes acidic due to its proton pump
3. A pH of 5.5 causes conformational changes in the viral hemagglutinin protein
4. Hemagglutinin anchors to the endosomal membrane
5. Brings viral and endosomal membrane together via another conformational change, causing their fusion
6. The proteins and nucleic acids are released and guided to the nucleus of the host cell and enter using host protein channels

42
Q

M2 ion channel

A

A pore in the influenza viral envelope. It pumps protons from the endosomal cavity into the interior of the virus. It also helps with the uncoating of the viral genome.

43
Q

Class 1 fusion proteins

A

Project vertically from the viral envelope. Look like spikes and are an present in trimeric form. They are mostly alpha helical. Found in HIV, influenza, Ebola, and others

44
Q

Class 2 fusion proteins

A

One example is flavivirus- Dengue and Zika. They are parallel to the membrane, mostly beta sheets, and are present as dimers. With the right trigger, they can become vertical and engage with the cell receptor.

45
Q

Fusion of Dengue Virus

A

The dengue virus attaches to the surface of a host cell and enters the cell by a process called endocytosis. Once deep inside the cell, where the environment is acidic, the virus fuses with the endosomal membrane and is released into the cytoplasm. The virus particle comes apart, releasing the viral genome.

46
Q

Entry of Ebola virus

A

Enters the cell via pinocytosis and then undergoes endocytosis in an endosome. NPC-1 is an endosomal receptor that mobilizes cholesterol, but is also needed for viral infection. It is bound by a viral glycoprotein on the viral envelope. The fusion peptide can then anchor to the endosomal membrane and the virus can be uncoated.

47
Q

Mutations in NPC-1

A

People with mutations in NPC-1 cannot be infected with Ebola virus. However, people with Newman-Pick disease cannot metabolize cholesterol and won’t live to adulthood

48
Q

Regulating factors of viral fusion (4)

A

Regulating factors are necessary so the virus uncoats at the correct place.
1. Receptor engagement
2. pH drop
3. Fusion peptide- they undergo proteolytic cleavage
4. Endosomal fusion receptor

49
Q

Uncoating of Adenovirus (7 steps)

A

Adenovirus is non-enveloped
1. The head of the viral fiber attaches to CAR (cell receptor)
2. Penton base interacts with integrins
3. The virus undergoes receptor
-mediated endocytosis, using clathrin-coated pits
4. Multiple uncoating steps where structural proteins are eliminated
5. Low pH causes release of penton base and facet
-stabilizing protein IX. Protein 9 is hydrophobic entering an aqueous environment- it shoots off and creates a hole in the endosome
6. Penton base mediates lysis of endosome membrane- the remainder of the virus is released to cytoplasm
7. Remaining capsid with viral DNA is transported using motor proteins (dynein) and docks onto nuclear pore complex

50
Q

Uncoating of poliovirus (6)

A

Poliovirus is non-enveloped and has an RNA genome
1. The virus binds to poliovirus receptor (also called CD155- on the cell surface)
2. The virus undergoes a conformational change and is internalized by the cell
3. Just below the cytoskeleton, the receptor binding to the capsid causes a pore to be opened in the capsid to release RNA
4. After the receptor binds to the canyons, the sequence of events facilitating the creation of the pore is begun. VP1, VP3, and VP4 are implicated in pore formation
5. The virus likely replicates in the cytoplasm
6. Movement is not dependent on endosomes, motor proteins, microtubules, or clathrin

51
Q

Types of uncoating strategies (3)

A
  1. Uncoating at the plasma membrane triggered by receptor engagement (like measles)
  2. Uncoating within endosomes
  3. Uncoating at the nuclear membrane
52
Q

Intracellular transport

A

Once in the cytoplasm, the genome must be transported to the nucleus or specific cytosolic membranes using different cellular machinery. This includes the cytoskeleton and cellular motor proteins

53
Q

Cellular motor proteins (2)

A
  1. Minus end directed microtubule-dependent motor (dynein) and adaptor protein (dynactin)
  2. Kinesins- transport cargo from (-) end to (+) end of microtubules
54
Q

Microtubules function

A

Made of 2 different types of proteins- beta tubulin and alpha tubulin. The two types of proteins alternate to form long fibers. Beta tubulin (- charge) is exposed on the cytoplasmic side and alpha tubulin (+ charge) is exposed on the nuclear side. With HIV, the reverse transcription complex is propelled along microtubules by dynein toward the microtubule - end. Reverse transcription likely happens inside the capsid

55
Q

Dynein function

A

Take cargo from + to - site of microtubules

56
Q

Kinesin function

A

Take cargo from - to + site of microtubules

57
Q

Nuclear import of viruses

A

Some viruses are transported on microtubules to the nucleus. This mostly includes large DNA viruses (HSV, adenovirus), but some RNA viruses (influenza A is the exception). Some DNA viruses will replicate in the cytoplasm. RNA viruses don’t need to go to the nucleus because they don’t need DNA processing machinery

58
Q

Models of nuclear import (4)

A
  1. Influenza A- endosome fuses with the viral envelope. The RNA is released, complexed with viral proteins
  2. HSV-1- the envelope fuses with the plasma membrane and releases the capsid. The capsid docks on the nuclear pore, releasing the DNA genome
  3. Adenovirus- the capsid is partially degraded, allowing the virus to dock on the nuclear pore. The DNA is released into the nucleus
  4. Parvovirus- these viruses accumulate on proteins outside the nuclear pore. This creates a new pore as the nuclear envelope degrades
59
Q

Nucleoproteins

A

Proteins that are complexed with viral nucleic acids, as found in influenza A. Guide the RNA genome through the nuclear pore. Nucleoproteins have certain signatures that help them to utilize the nuclear import system, and helps them to utilize the host machinery

60
Q

What facilitates the entry of the nuclear genome?

A

For many viruses, the entire capsid doesn’t need to enter the nucleus. However, virally encoded proteins need to complex with the genome so it can enter the nucleus. For targeting to the nucleus, viruses use nuclear localization signals (NLS)

61
Q

Nuclear localization signals (NLS)

A

An amino acid sequence that targets viral proteins to the nucleus. Any protein going from the cytoplasm to the nucleus should have this specific sequence. Most proteins that complex with viral nucleic acid also have this sequence. Importins are one example of proteins with an NLS. HIV-1 and adenovirus have importin 7, and influenza virus nucleoproteins have importins alpha and beta

62
Q

Types of nuclear localization signals (2)

A
  1. Simple- has 3-7 basic amino acids flanked by hydrophobic amino acids
  2. Bipartite- 2 basic amino acid regions are separated by random amino acids
63
Q

Steps involved in nuclear transport (4)

A
  1. Importin alpha binds to cargo, and Importin beta binds to importin alpha
  2. This complex binds to the nucleoporin complex on the nuclear pore
  3. Ran-GDP protein joins the cargo complex and allows the complex to enter through the nuclear pore
  4. Ran-GDP is phosphorylated to become Ran-GTP. This drives dissociation of the cargo so it can stay in the nucleus
64
Q

Direct cell-to-cell transfer

A

Viruses can be transmitted directly from cell-to-cell (only demonstrated in enveloped viruses). It has been demonstrated in measles, vaccinia, and HIV and other lentiviruses

65
Q

Measles cell-to-cell transfer

A

Fusogenic envelope proteins induce fusion of infected cells with neighboring uninfected cells. Koplick spots are actually fused together cells due to measles

66
Q

Vaccinia cell-to-cell transfer

A

Virus particles are pushed into neighboring cells by localized actin polymerization inside of the infected cell

67
Q

HIV cell-to-cell transfer

A

Virological synapses are important for spread between interacting immune cells

68
Q

Virological synapses in HIV infection

A

HIV may form polysynapses and filopodial bridges that allow for passage of virus particles without engaging the receptor. This may allow the virus to result in rapid viremia, which can cause severe infection

Viral Pathogenesis (9 decks)

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