Virus Replication in Host Cells

Question 1

Introduction to virus replication

Answer 1

• The intention of every virus is to identify and invade a suitable host cell for replication or multiplication of its gene.
• After replication, the infected host cell is lysed to release copies of the virus genome which invade new hosts.
• Most viruses are host and tissue specific (tissue tropism). E.gHepatitis C only affects the liver cells

Question 2

Key Viral Components

Answer 2

Viruses are made up of two key components:

1. Genetic material: Viruses are made up either DNA or RNA, but never both.No cellular structure present (acellular)
2. Capsid: Layer of protein surrounding the genetic material of a virus. Capsids are made up of smaller units called Capsomeres.
3. Envelope: Extra layer of lipoprotein covering the capsid. Not present in all viruses

Question 3

Role of the Capsid

Answer 3

Capsid protects the nucleic acid when teh virus is outside the host cell

Helps binds to a suitable host cell surface receptors using spike proteins on an envelop (if present) or transfer it’s genetic material into host cell through endocytosis if its a naked virus (that is no envelop)

e.g. Poxvirus (complex virus) lacks a typical capsid and are covered by a dense layer of lipoprotein

Question 4

Roles of a genetic material

Answer 4

Carries gene: necessary to invade host cells.

Note the the number of genes varies for each virus (few to hundreds)

Redirect or manipulate host cell machinery into producing viral cells.

Viruses lack protein synthesizing machinery: However, they contain parts needed to evade host cells and manipulate the cell replicating machinery into producing more copies of the virus rather than normal cellular protiens.

Question 5

Steps in virus genome replication

Answer 5

1. Attachment of virionto suitable host cell.
2. Entry of virus into the cell
3. Transcription of virus genes into mRNA (messenger RNA) molecules.
4. Translation of virus mRNA into virus proteins.
5. Genome replication
6. Assembly of virus proteins and genomes into new virions.
7. Exit of virionsfrom host infected cells.

Note: not all viruses undergo all seven steps above, some viruses may need extra steps while some steps may occur concurrently in some viruses in which case steps 3-7 may occur at the same time

Question 6

Attachment of viruses to host cells

Answer 6

• Viruses that infect animals and bacteria must first attach themselves to host cell surface barriers (cell wall for bacteria or cell membrane for animals)
• Cell membrane: Animal cells possess a cell membrane which the virus must attach itself to via specific cell surface receptors in order to invade the cell.
• Cell wall: Likewise, bacteria cells possess a cell wall which the virus must attach itself to via specific cell surface receptors in order to invade the cell.
• Note that virus attachment and entry in plants cell are mostly mediated by vectors.

Question 7

Attachment of Animal viruses via cell receptors

Answer 7

• Host cell surface receptors are proteins which a virus can bind using its attachment structures prior to cell entry.
• Binding of cell surface receptors and virus attachment structures are specific like a ‘’Lock and key’’.
• Sometimes a virus may need to bind to a second host cell receptor known as a co-receptorprior to binding.
• Cell receptors and co-receptors function to mediate cell-to-cell contact and binding as well as receptors for chemokinesand growth factors.
• Binding of cell surface receptors and virus attachment structures result in a conformational change in virus proteins which initiates the binding.
• Most host cell surface receptors used by viruses contain sugar molecules (glycoproteins) composed of folded domains similar to immunoglobulin

Question 8

Virus attachment sites

Answer 8

•Viruses possess multiple binding sites on their surfaces which is largely dependent on the absence (naked viruses) or presence of an envel

Question 9

Virus attachment sites

Naked Viruses

Answer 9

• Nakedviruses: The attachment sites for naked viruses are on the capsid which differ in topology and may be in the form of ridges (foot and mouth disease virus), or within depressions (poliovirus). Both belong to the picornavirusgroup of viruses.
• Also, attachment sites for some naked viruses could be on specialized structures such as spikes of rotaviruses, or on fibers and knobs of adenoviruses.
• Binding of picornavirusesto host cell receptors result in major structural changes in the virion

Question 10

Virus attachment sites

Enveloped viruses

Answer 10

•Enveloped viruses: Host cell attachment structures for enveloped viruses are on glycoproteins present on the envelope.

Question 11

Virus attachment sites

Haemagglutinins

Answer 11

• Haemagglutinins: Some virionspossess proteins which binds to immunoglobulin causing them to clump (haemagglutination). E.g. are measles virus and influenza virus.
• Forces involved in virus-host cell binding are weak forces involving hydrogen bonding, van der Waals forces and ionic forces.
• Sugar molecules on host cell receptors and /or on the virionare involved in the binding forces
• Initial binding of viruses to host cells involves weak forces which are reversible, which become irreversible with binding of more recepto

Question 12

Step 2. Entry of viruses into animal cel

Answer 12

• After attachment, a virus must gain entrance into host cells for replication.
• Entry: a virus must gain entry into host cells either through the cell surface or through the an endosome membrane (tiny vesicle forming part of a plasma membrane which break offs into the cytoplasm) in a process called endocytosis.
• Endocytosis: Aprocess which serve several roles for cells including nutrients uptake and defense against pathogens is hijacked by viruses to gain entry into cells

Question 13

Entry of viruses into animal cells (Endocytosis

Answer 13

•Animal viruses gain entry into host cells through two main endocyticmechanisms:

1. Clathrin-mediated endocytosis: Clathrinis a cell protein found around the inner side of cell membrane. Clathrinforms a coat around viruses resulting in pit and invagination of the cell membrane which is budded offand shed allowing the virus entry into cell. Virus examples are the adenoviruses and vesicular stomatitis virus.
2. Caveolin-mediated endocytosis: Similarly viruses such as simian virus 40 coat themselves with cell membrane protein (caveolin) allowing them entry into cells through endocytosis

Question 14

Naked virus entry into animal cells (Endocytosis

Answer 14

•Naked viruses attach and release their genome through a pore in the cell membrane by endocytosis

Question 15

Enveloped virus entry into animal cells (Endocytosi

Answer 15

•Host cell entry may occur either by

1. Endocytosis or fusion of virus envelop containing glycoprotein with plasma membrane
2. Fusion of virus envelop with the cell endosome membrane.Virus fusion proteins are hiddenin the envelop and are released upon the binding of a virus to a host cell receptor resulting in a series of conformational changes.The changes fuse the virus envelop and cell membrane first from the outer layers in a process called hemifusion, followed by fusion of the inner layers, completing the fusion proces

Question 16

Intracellular transport of cell-invading viru

Answer 16

• Microtubules: One of the cell transport systems exploited by viruses to arrive at the nucleus.
• Most RNA viruses replicate in the cytoplasm.
• Exceptions are:

1. Retroviruses: They copy their RNA genome into DNA in the cytoplasm which is then transported to the nucleus during cell division (mitosis) for replication.
2. Influenza viruses: require the cell splicing machinery in the nucleus to get rid of intervening sequences (introns).

Note that the lentiviruses, e.g. HIV can transport its DNA from the cytoplasm into the intact nucleus with cell division in progress.

• Most DNA viruses can only replicate their genome in the nucleus and so utilize the microtubule as a transport to the nucleus before entry through the nuclear pores (parvoviruses, herpesviruses, retroviruses and adenoviruses).
• Exemptions are poxviruses and iridoviruses: replicate their DNA in the cytoplasm

Question 17

Uncoating of virus genome

Answer 17

• Viruses shed their capsid on entry into a host cell releasing its genome.
• For animal viruses, uncoatingcan occur either in the cytoplasm, nuclear pore or in the nucleus.
• Virus gene replication may not occur immediately upon successful virus-cell entry.
• Host intracellular defenses such as lysosomalenzymes may neutralize infectivity before or after virus uncoating.
• In certain cases, invading viruses may initiate a latent infection rather than begin a complete replication cycle.
• Also, under favourablecell conditions (that is virus survives and in right cell), gene replication involving transcription can commence

Question 18

Host cell entry by bacteriophages

Answer 18

• Phages like animal viruses attach to host cell through specific surface molecules (receptors) on its cell wall.
• Most phages inject their genome into the host cell leaving behind its capsid on the cell wall. This is in contrast to animal viruses.
• Phage enzymes (e.g. lysozymes): virionsof many phages carry lysozymes to aid genome injection through the cell wall.
• Mycoplasma: Are exemptions as they lack a cell wall.

Question 19

Four classes of viral genomes

Answer 19

5. A virus genome is either DNA or RNA. Different from cellular organisms whose genetic unit is a DNA.

•Viruses can be divided into four groups of based on the number of strand in its genome.

1. double-stranded DNA (dsDNA)
2. single-stranded DNA (ssDNA)
3. single-stranded RNA (ssRNA)
4. double-stranded RNA (dsRNA)

Question 20

Viral genome replication

slide 21, class 4

Answer 20

Genome replication of some viruses somewhat aligns with the universal rule of DNA replication in molecular biology with some exemptions.

Figure 5: Central dogma in Molecular biology and virus modifications

Question 21

Figure 4: Baltimoremethod for virus classification:

see picture, slide 22, class 4

Answer 21

classification:Strands of virus nucleic acid are either labeled as +veor –vedepending on their identity with virus mRNA.

The strand with the same sequence as the virus mRNA is termed +ve, while the strand complementary to virus mRNA is termed -ve.
Note the following:

class I(dsDNA) and class iii(dsRNA) are transcribed directly to virus mRNA.

Class IIhas single stranded DNA (ssDNA) which is either +ssDNAor –ssDNA. Both are complementary to each other resulting in a dsDNAwhich is transcribed to mRNA.

Class IVhas single stranded +RNA (+ssRNA) which give rise to complementary (–)RNA which in turn gives rise to mRNA

Class Vhas single stranded-ssRNAresults in a +RNA which is transcribed to mRNA

Class VI: + ssRNAcan be reversed transcribed to -ssDNA, which produces the complementary +ssDNA, both combined to be dsDNAwhich is transcribed into mRNAClass VII: dsDNAcan transcribe directly to mRNA or give rise to (+) RNA which is reversed transcribed to (-) DNA which produced its complementary +DNA resulting in dsDNA

Question 22

look at slide 23, class 4

Answer 22

figure 6:

Schematic diagram showing transcription and translation in an eukaryotic cell, and prokaryotic cell

Question 23

Viruses and transcriptases

Answer 23

Generally, DNA viruses which transcribe their genes into mRNA in the nucleus utilize host cell enzymes.The retroviruses are included as they copy their RNA genome into DNA in the cytoplasm which gains entry to the nucleus for transcription

Most virus that encode their own transcriptase possess the enzyme except the plus-strand RNA viruses which synthesize the enzyme

Question 24

Viruses and transcriptases

DNA viruses

Answer 24

DNA viruses which transcribe their gene in the cytoplasm encode their own transcriptasesmainly due to absence of this enzyme in the cytoplas

Question 25

Viruses and transcriptases Rna Viruses

Answer 25

Most RNA viruses transcribe their gene in the cytoplasm where cell RNA transcriptasesare found (RNA-dependent RNA polymerase

Question 26

Viruses and transcriptases Class III, IV and V

Answer 26

Viruses in class III, IV and V (RNA viruses) encode their own transcriptase enzymes despite the presence of cell ssRNA-dependent RNA polymerases

Question 27

Viruses and transcriptases Group VI and VII Reverse Transcriptases

Answer 27

Reverse transcriptases(group VI and VII): Copies RNA and DNA templates into DNA using RNA –dependent DNA polymerase and DNA-dependent DNA polymerase respectively

Question 28

Overview of virus genome replication

Answer 28

•Virus genome replication is the fifth step in replication cycle in which an invading virus makes copies of its genes for progeny offspring .•Most DNA viruses copy their genes directly to DNA * Most RNA viruses copy their genes directly to RNA * A few DNA virus replicate their genes through RNA intermediate * A few RNA virus replicate their genome through DNA intermediate

Question 29

Figure 8: Virus genome replication based on Baltimore classification:

Answer 29

Strands of virus nucleic acid are either labeled as +veor –vedepending on their identity with virus mRNA.The strand with the same sequence as the virus mRNA is termed +ve(except that in DNA thymine replaces uracil), while the strand complementary to virus mRNA is termed -ve.Single-stranded DNA is converted to dsDNAprior to copying

Question 30

Replication of viruses in eukaryotic cells

Answer 30

•Eukaryotic viruses upon cell entry either replicate their genome in the cytoplasm or transported into the nucleus. Destination is dependent on the type of genome

Question 31

Replication of viruses in eukaryotic cell Replication of viruses in eukaryotic cell

Answer 31

Note:The genome of most DNAviruses are replicated in the nucleus; but those of some dsDNAare replicated in the cytoplasm. Most RNAviruses replicate their genome in the cytoplasm with exemption of some minus-strand RNA viruses. The reverse transcriptase viruses (retroviruses and pararetroviruses) each replicates RNA to DNA in the cytoplasm

Question 32

Replication of DNA viruses

Answer 32

* Class I (dsDNA) and class II (ssDNA) viruses replicate their genome via dsDNA * ssDNAsynthesizes its complimentary strand to become dsDNA. * Each viral DNA has at least one specific sequence where replication is initiated (replication origin). * Proteins involved in DNA replication binds to this site and they include: 1. A helicase (unwinds the double helix at that site) 2. A ssDNAbinding protein (keeps the two strands apart) 3. A DNA polymerase

Question 33

Replication of Double-stranded RNA viruses

Answer 33

* dsRNAreplication is similar to dsDNAin that the double strand must be unwound by helicase before replication can occur. * Some dsRNAviruses replicate their genome by two mechanisms: 1. Semi-conservative mechanism: Some dsRNAviruses replicate their genome similar to dsDNAwhereby each double stranded progeny molecule is made up of the parent strand and a daughter strand (Pseudomonas phage phi 6). 2. Conserved mechanism: Some dsRNAviruses replicate their genome in a process whereby the double stranded molecule of the infecting virus genome is conserve

Question 34

Replication of Double-stranded RNA viruse

Answer 34

Figure 12: Conservative and semi-conservative replication

Question 35

Replication of single-stranded RNA viruses

Answer 35

* Viruses with ssRNAin class IV (+ssRNA) and class V (-SSRNA) replicate their genome by synthesizing their complimentary strands. * Synthesis of each RNA molecule requires the RNA-dependent RNA polymerase at the 3’ end of the template. * Plus-strand RNA viruses of eukaryotes replicate their RNA in association with membranes derived from the cytoplasmic membranous structures. * Minus-strand RNA replicate by coating their RNA template with viral protein and not host membrane

Question 36

Replication of reverse transcriptase viruses

Answer 36

* Some RNA viruses replicate their genes through DNA intermediates. * Some DNA viruses replicate their genome through RNA intermediate. * Both virus groups utilize the reverse transcriptase enzymes. * Reverse transcription occurs within the viral structure in the cytoplasm of host cel