Chapter 6

An Introduction to Viruses

Introduction

All life-forms can be infected by viruses.

Some viruses generate serious epidemics, from dengue fever to influenza to AIDS.

Others fill essential niches in the environment, particularly in marine ecosystems.

In research, viruses have provided both tools and model systems in molecular biology.

This 11-inch-high limestone Egyptian funerary stele is from Saqqara, 10 miles south of Cairo; Amarna Period, 18th Dynasty (1403-1365 BCE), Glyptotek Museum, Copenhagen. The stele portrays Roma (or Rema), an Egyptian doorkeeper, and his family giving offerings to the Goddess Astarte. Thought to be the earliest depiction of a victim of poliomyelitis, the man adeptly carries a goblet while supporting himself with a staff. His withered right leg and deformed right foot are characteristic of poliomyelitis.

Ramses V, Pharaoh of Egypt He died ~1145 BCE, presumably of smallpox. His mummified head and torso bear the characteristic lesions of the disease. Smallpox victims included many other rulers throughout history, among them Louis XV of France, Mary II of England, and the Holy Roman Emperor Joseph I.

The search for the elusive virus

Louis Pasteur postulated that rabies was caused by a virus (1884)

Ivanovski and Beijerinck showed a disease in tobacco was caused by a virus (1890s)

Viruses: non-cellular particles with a definite size, shape, and chemical composition

Viral diseases led to the development of some of the first vaccines.

Poliovirus causes poliomyelitis, which can lead to paralysis.

President Franklin Roosevelt established the March of Dimes.

With its support, Jonas Salk developed the first polio vaccine in 1952.

What Is a Virus?

A virus is a non-cellular particle that must infect a host cell (obligate intracellular parasite), where it reproduces.

It typically subverts the cell’s machinery and directs it to produce viral particles.

The virus particle, or virion, consists of a single nucleic acid (DNA or RNA) contained within a protective protein capsid.

The position of viruses in the biological spectrum

There is no universal agreement on how and when viruses originated

Viruses are considered the most abundant microbes on earth

Viruses played a role in the evolution of Bacteria, Archaea, and Eukarya

Viruses are obligate intracellular parasites

Viruses are ubiquitous in all environments and part of our daily lives

Most frequent infections of college students:

1) Respiratory pathogens such as rhinovirus (the common cold) and Epstein-Barr virus (infectious mononucleosis)

2) Sexually transmitted viruses such as herpes simplex virus (HSV) and papillomavirus (genital warts)

Different viruses infect every group of organisms

Each species of virus infects a particular group of host species, or host range

Viruses infect all forms of life

Viroids are RNA molecules that infect plants.

They have no protein capsid.

Are replicated by host RNA polymerase.

Some have catalytic ability.

Viroids

Prions are proteins that infect animals.

They have no nucleic acid component.

Have an abnormal structure that alters the conformation of other normal proteins

Extremely resistant to usual sterilization techniques

Prions

Prions Diseases

Common in animals

Scrapie in sheep and goats

Bovine spongiform encephalopathies (BSE), a.k.a. mad cow disease

- transmissible and fatal neurodegenerative disease

Humans – Creutzfeldt-Jakob Syndrome (CJS)

Viral structure

Viruses bear no resemblance to cells

Lack protein-synthesizing machinery

Viruses contain only the parts needed to invade and control a host cell

Virus structure

The viral capsid is the protein shell of a virus.

The capsid encloses the viral genome

The capsid delivers the viral genome into the host cell.

Different viruses make different capsid forms.

Capsids

All viruses have capsids (protein coats that enclose and protect their nucleic acid)

The capsid together with the nucleic acid is the nucleocapsid

Each capsid is made of identical protein subunits called capsomers

Some viruses have an external covering called an envelope; those lacking an envelope are naked

Helical – Rod or thread-like continuous helix of capsomers forming a cylindrical nucleocapsid

Structural types of capsids

Icosahedral - Polyhedral with 20 identical triangular faces

Have a structure that exhibits rotational symmetry

Structural types of capsids

In some icosahedral viruses, the capsid is enclosed in an envelope, formed from the cell membrane.

The envelope contains glycoprotein spikes, which are encoded by the virus. Spikes are essential for attachment of the virus to the host cell

Between the envelope and capsid, tegument proteins may be found.

Viruses lacking an envelope are naked

Envelope

Dr.Stepehen Fuller

Functions of capsid

1. Protect genome from atmosphere (May include damaging UV-light, shearing forces, nucleases either leaked or secreted by cells). 2. Virus-attachment protein- interacts with cellular receptor to initiate infection. 3. Delivery of genome in infectious form. May simply “dump” genome into cytoplasm (most +ssRNA viruses) or serve as the core for replication (retroviruses and rotaviruses).

These have complex multipart structures

T4 bacteriophages: Have an icosahedral “head” and helical “neck”

Poxviruses lack a typical capsid and are covered by a dense layer of lipoproteins

Viruses with unusual structures

Filamentous viruses

The capsid consists of a long tube of protein, with the genome coiled inside

Vary in length, depending on genome size

Include bacteriophages (e.g. M13) as well as animal viruses (Ebola)

Bacteriophage M13

Viruses with unusual structures

Ebola virus

Bacteriophage

Bacterial viruses (phages)

Most widely studied are those that infect Escherichia coli – complex structure, DNA Are called T-even phages as T2, T4 & T6 of the 7 phages studied have similar structures

Types of viruses

Nucleic acids

Viral genome – either DNA or RNA but never both

Carries genes necessary to invade host cell and redirect cell’s activity to make new viruses

Number of genes varies for each type of virus – few to hundreds

Nucleic acids

• DNA viruses

Usually double stranded (ds) but may be single stranded (ss)

Circular or linear

• RNA viruses

Usually single stranded, may be double stranded, may be segmented into separate RNA pieces

RNA genomes ready for immediate translation are positive-sense RNA

RNA genomes that must be converted into proper form are negative-sense RNA

Pre-formed enzymes may be present

– Polymerases – DNA or RNA

– Replicases – copy RNA

– Reverse transcriptase – synthesis of DNA from RNA (HIV-1)

Viral enzymes

Viral replication- rules

All viruses require a host cell for reproduction.

Thus, they all face the same needs for host infection:

Host recognition and attachment

Genome entry

Assembly of progeny virions

Exit and transmission

Replication cycle of Bacteriophages

Multiplication goes through similar stages as animal viruses

Only the nucleic acid enters the cytoplasm - uncoating is not necessary (Hershey- Chase experiment)

Release is a result of cell lysis induced by viral enzymes and accumulation of viruses - lytic cycle

Hershey- Chase experiment

http://scarc.library.oregonstate.edu/coll/pauling/dna/index.html

Steps in phage replication

1. Adsorption – binding of virus to specific molecules on host cell

2. Penetration – genome enters host cell

3. Replication – viral components are produced

4. Assembly – viral components are assembled

5. Maturation – completion of viral formation

6. Lysis & Release – viruses leave the cell to infect other cells

Contact and attachment are mediated by cell-surface receptors.

Proteins that are specific to the host species and which bind to a specific viral component.

Bacterial cell receptors are normally used for important functions for the host cell. Example: sugar uptake

Bacteriophages attach to host cells

Bacteriophages (phages) inject only their genome into a cell through the cell envelope.

The phage capsid remains outside, attached to the cell surface (ghost).

Phage reproduction within host cells

Orlova EV. How viruses infect bacteria? The EMBO Journal 2009;28(7):797-798. doi:10.1038/emboj.2009.71.

1) Lytic cycle

Bacteriophage quickly replicates, killing host cell

2) Lysogenic cycle

Bacteriophage is quiescent.

Integrates into cell chromosome, as a prophage.

Can reactivate to become lytic.

The “decision” between the two cycles is dictated by environmental cues

In general, events that threaten host cell survival trigger a lytic burst

Bacteriophages can undergo two different types of life cycles

Lysogeny: The silent virus infection

• Not all phages complete the lytic cycle

• Some DNA phages, called temperate phages, undergo adsorption and penetration but don’t replicate

• The viral genome inserts into bacterial genome and becomes an inactive prophage – the cell is not lysed

• Prophage is retained and copied during normal cell division resulting in the transfer of temperate phage genome to all host cell progeny – lysogeny

• Induction can occur resulting in activation of lysogenic prophage followed by viral replication and cell lysis

Lysogeny

• Lysogeny results in the spread of the virus without killing the host cell

• Phage genes in the bacterial chromosome can cause the production of toxins or enzymes that cause pathology – lysogenic conversion

– Corynebacterium diphtheriae

– Vibrio cholerae

– Clostridium botulinum

The primary factor determining the life cycle of an animal virus is the form of its genome.

DNA viruses

Can utilize the host replication machinery

RNA viruses

Use an RNA-dependent RNA-polymerase to transcribe their mRNA

Retroviruses

Use a reverse transcriptase to copy their genomic (RNA) sequence into DNA for insertion in the host chromosome

Animal Virus replication cycles

Modes of animal viral multiplication

General phases in animal virus multiplication cycle:

1.Adsorption – binding of virus to specific molecules on the host cell

2.Penetration – genome enters the host cell

3.Uncoating – the viral nucleic acid is released from the capsid

4.Synthesis – viral components are produced

5.Assembly – new viral particles are constructed

6.Release – assembled viruses are released by budding (exocytosis) or cell lysis

Animal viruses bind specific receptor proteins on their host cell.

Receptors determine the viral tropism.

Ebola virus exhibits broad tropism, infecting many kinds of host tissues.

Papillomavirus shows tropism for only epithelial tissues.

Most animal viruses enter host as virions.

Internalized virions undergo uncoating, where genome is released from its capsid.

Animal viruses show tissue tropism

Envelope spike

Host cell membrane

Receptor

Receptor

Capsid spike

Host cell

membrane

Adsorption and host range

• Virus coincidentally collides with a susceptible host cell and adsorbs specifically to receptor sites on the membrane

• Spectrum of cells a virus can infect – host range

– Hepatitis B – human liver cells

– Poliovirus – primate intestinal and nerve cells

– Rabies – various cells of many mammals

Penetration/Uncoating

• Flexible cell membrane is penetrated by the whole virus by:

– Endocytosis – entire virus is engulfed and enclosed in a vacuole or vesicle

– Fusion – envelope merges directly with membrane resulting in nucleocapsid’s entry into cytoplasm

Variety in penetration and uncoating

Replication and protein production

• Varies depending on whether the virus is a DNA or RNA virus

• DNA viruses generally replicate and assemble in the nucleus

• RNA viruses generally replicate and assemble in the cytoplasm

– Positive-sense RNA contain the message for translation

– Negative-sense RNA must be converted into positive-sense message

Papillomavirus (DNA) Life Cycle

HPV, a double-stranded DNA virus, enters the cytoplasm, where the protein coat disintegrates. The viral DNA enters the nucleus for replication and transcription by host polymerases. Viral mRNA returns to the cytoplasm for translation of capsid proteins, which return to the nucleus for assembly of virions.

Picornavirus (RNA) Life Cycle

Picornavirus life cycle. A picornavirus inserts its (+) strand RNA into the cell. Reproduction occurs entirely in the cytoplasm. A key step is the early translation of a viral gene to make RNA-dependent RNA polymerase. The polymerase uses the picornavirus RNA template to make (–) strand RNA, which then serves as a template for other viral mRNAs, as well as progeny genomic RNA.

HIV (Retrovirus) Life Cycle

Retrovirus life cycle. A retrovirus such as human immunodeficiency virus (HIV) uses reverse transcriptase to copy its RNA into double-stranded DNA. The DNA then enters the nucleus to integrate in the host genome. Host RNA polymerase generates viral mRNA and viral genomic RNA. The viral mRNA enters the cytoplasm for translation. Virions assemble near cell membrane and bud out.

Release

• Assembled viruses leave the host cell in one of two ways:

– Budding – exocytosis; nucleocapsid binds to membrane which pinches off and sheds the viruses gradually; cell is not immediately destroyed

– Lysis – nonenveloped and complex viruses released when cell dies and ruptures

Late Uta von Schwedler

Damage to host cell

Cytopathic effects - virus-induced damage to cells

1. Changes in size and shape

2. Cytoplasmic inclusion bodies

3. Inclusion bodies

4. Cells fuse to form multinucleated cells

5. Cell lysis

6. Alter DNA

7. Transform cells into cancerous cells

Effects of some human viruses

Persistent infections

Persistent infections - cell harbors the virus and is not immediately lysed

Can last weeks or host’s lifetime; several can periodically reactivate – chronic latent state

Measles virus – may remain hidden in brain cells for many years

Herpes simplex virus – cold sores and genital herpes

Herpes zoster virus – chickenpox and shingles

• Some animal viruses enter the host cell and permanently alter its genetic material resulting in cancer – transformation of the cell

• Transformed cells have an increased rate of growth, alterations in chromosomes, and the capacity to divide for indefinite time periods resulting in tumors

• Mammalian viruses capable of initiating tumors are called oncoviruses or oncogenic viruses

– Papillomavirus – cervical cancer

– Epstein-Barr virus – Burkitt’s lymphoma

– Hepatitis C virus – Liver cancer

Viral damage

Dependent on other viruses for replication

Adeno-associated virus – replicates only in cells infected with adenovirus

Delta agent – naked strand of RNA expressed only in the presence of hepatitis B virus

Satellite viruses

Oncogenic viruses transform the host cell to become cancerous.

Mechanisms of oncogenesis include:

1) Insertion of an oncogene into the host genome

2) Integration of the entire viral genome

3) Expression of viral proteins that interfere with host cell cycle regulation

Oncogenic viruses

Techniques in cultivating and identifying animal viruses

Methods used:

– Cell (tissue) cultures – cultured cells grow in sheets that support viral replication and permit observation for cytopathic effects

– Bird embryos – incubating egg is an ideal system; virus is injected through the shell

– Live animal inoculation – occasionally used when necessary

Methods for growing viruses

Animal viruses can be cultured within whole animals by serial inoculation

Ensures that the virus strain maintains its original virulence, but process is expensive and laborious.

They can also be grown in human cell tissue culture.

Tissue culture of animal viruses

Plaque assay of bacteriophages

Plaque assay of animal viruses

Medical importance of viruses

• Viruses are the most common cause of acute infections

• Several billion viral infections per year

• Some viruses have high mortality rates

• Possible connection of viruses to chronic afflictions of unknown cause

• Viruses are major participants in the earth’s ecosystem

Detection and treatment of animal viral infections

• More difficult than other agents

• Consider overall clinical picture

• Take appropriate sample

– Infect cell culture – look for characteristic cytopathic effects

– Screen for parts of the virus

– Screen for immune response to virus (antibodies)

• Antiviral drugs can cause serious side effects

Other noncellular infectious agents

Satellite viruses – dependent on other viruses for replication

Adeno-associated virus – replicates only in cells infected with adenovirus

Delta agent – naked strand of RNA expressed only in the presence of hepatitis B virus