Viruses and Viral Replication

History of Virology

     The concept that agents of infection and disease could be smaller than even bacterial cells was unknown until the late nineteenth century.  In 1884, Charles Chamberland, the inventor of the autoclave, developed filters which could trap bacteria while allowing the fluid they were suspended in to pass through.  He believed that this would produce a sterile medium without heating.  However, two years earlier, Dimitri Ivanowski had reported that the cause of the disease tobacco mosaic was filterable, meaning that it could pass through the filters of his day, yet still cause the disease when placed on healthy plant leaves.  In 1898, Martinus Beijerink would, without knowledge of Ivanowski's work, report the same conclusions, referring to tobacco mosaic as a "contagious living liquid.  In the same year, Friedrich Loeffler and Paul Frosch suggested that the agent of foot and mouth disease, as well as many other pathogens were filterable.  Later, Peyton Rous demonstrated that filtered fluid from animal tumor cells could cause healthy cells to become tumors as well, and  Walter Reed found that the agent of yellow fever was also viral, and was carried by a mosquito which serves as its vector.

     One the major problems which had to be overcome in order to fully understand the nature of viruses was to find an efficient means to cultivate these infective agents.  While bacteria could be grown easily on artificial media, viruses could not.  Viral particles appeared as crystaline when purified, so their exact nature was unknown, and the name contagium vivum fluidum (contagious living fluid) was proposed.  Two French scientists, Frederick Twort and Felix d'Herelle separately discovered that some viruses called bacteriophages, could infect and destroy bacterial cells, and two Americans, Robert Nye and Frederick Parker found that they could successfully raise animal viruses on cultures produced by cultivating eukaryote cells in artificial media.  Such cell cultures could be used to study viruses of many types.  Other workers would discover that some viruses could be cultured in chicken eggs (Goodpasture et al., 1931), bacteriophages pass DNA to their host cells (Hershey and Chase, 1952), and tobacco mosaic virus passes RNA to the host cell it infects (Fraenkel-Conrat et al., 1957).  All of these discoveries, as well as those of other workers, would enhance our understanding of viruses and ultimately lead to the development of vaccines against many viral diseases, specific in-vitro tests to determine the presence of viruses in the body of an infected individual, and the synthesis of synthetic antiviral agents used to combat such diseases as herpes, influenza, and AIDS.

Characteristics of Viruses

     Viruses have some of the characteristics of living things, i.e. biologically important molecules, proteins and nucleic acids, as well as the ability to replicate.  However, viruses are not cells.  They can only reproduce while in the cytoplasm of a host cell by preempting normal cell activities and forcing the cell to biosynthesize new viral particles.  As such, they are called obligate intracellular parasites.  Since viruses cannot actively metabolize and are inert unless in the presence of host cells, many regard them as nonliving extensions of the host.  Their status is still debated today, so perhaps it is best to refer to viruses as acellular biological entities.

    All viruses have two major components, a protein coat called a capsid, which is composed of individual protein subunits called capsomeres, and a nucleic acid core composed of either DNA or RNA, but not of  both together.  The combination of the capsid and nucleic acid core is called the nucleocapsid.  Some viruses, such as influenza and HIV, have an external viral envelope, which is composed of cell membrane modified by the virus prior to release from the host cell.  This envelope is responsible for the pathogenicity of the virus it surrounds, since it is permiated by protein "spikes" which serve as sites which bond to receptors on the cell membrane of potential new host cells.  Nonenveloped viruses, such as the adenovirus, carry their protein spikes on the capsid itself.

     Since the envelope is composed mostly of nonpolar lipids, it can be removed by contact with the nonpolar solvent ether.  Enveloped viruses are thus referred to as ether soluble, since they can be inactivated, or attenuated in this way.  Attenuated viruses can still elicit the production of antibodies by a host organism, so they can be used to produce vaccines against those viruses of the same type which still retain their envelope.  Nonenveloped forms, however, are not inactivated by ether, so they still remain infective.

Viral Replication

    Viruses can only replicate by utilizing the biosynthetic capabilities of the host cell.  Normally, we can think of the viral replication cycle as having four major steps:

1. Attachment (Adsoption)- the virus attaches itself  using either its tail fibers (bacteriophage) or
    protein "spikes" which bind to receptor-sites on the surface of the host cell wall or membrane.
    Viruses are nonmotile, thus attachment depends on random brownian motion to bring the virus
    into contact with the potential host cell.

2. Penetration and Uncoating- Once the virus has attached itself, it can then penetrate the host cell
    wall or membrane.  The protein coat is not necessary for viral replication and must be removed
    through the process called uncoating.  Uncoating occurs along with attachment in bacteriophage
    replication, since the bacteriophage "injects" its nucleic acid into the host cell.  The empty protein
    coat then will either remain attached to the host cell, or will fall off.  In viruses which parasitize
    eukaryotic cells, the process of uncoating depends upon the type of  virus.  Some nonenveloped
    viruses such as the adenovirus simply attach, then physically force their nucleic acid into the cell,
    while others trigger the host to pull them in via phagocytosis.  Enveloped viruses incorporate their
    envelope with the cell membrane of the host, allowing the virus to enter and be enzymatically
    stripped of its protein coat.

     Once uncoating has occurred, the nucleic acid of the virus remains in the cell for a period of time known as the eclipse phase.  During this period, it is physically impossible to tell that the cell has actually been infected.  Some varieties of virus have or produce DNA which incorporates with the genome of the host cell to become a prophage (bacteriophage) or provirus (eukaryotic viruses).  This set of viral genes may remain untranscribed for long periods of time, during which the cell continues to live and metabolize normally, even to replicate and give rise to new viral infected cells.  When this occurs, the viral cycle is said to have become temperate, or to have entered the temperate or lysogenic phase.  If viral DNA is incorporated into the chromosome of some bacteria, these can gain the ability to become pathogenic (i.e. to switch on latent genes which encode for pathogenic properties such as the production of toxins).  This phenomenon is called lysogenic conversion, and can occur in such bacterial genera as Staphylococcus, Bacillus, Haemophilus, and Clostridium.

3.  Maturation and Assembly- When the viral nucleic acid does begin to express itself, either
     through the activity of viral enzymes, or transcription of incorporated viral DNA, the normal
     metabolic activities of the cell cease, and the cell's own biosynthetic processes are used in the
     production of early proteins, which are subunits of the virus protein coat and/or portions of the
     viral envelope, as well as new viral nucleic acids (either DNA or RNA, depending upon the type
     of virus infecting the cell).  Early  proteins are assembled inside the cell, becoming late proteins,
     which surround the new viral nucleic acids.  While this maturation process is occurring, clusters
     of new virions, called inclusion bodies, become visible within the infected cell.  Inclusion
     bodies  (example- Cowdry bodies in cells infected with the rabies virus), malformed cells, and
     damaged or destroyed cells are called cytopathic effects.

4.  Release-  In the cycle of the bacteriophage and many of the eukaryote-infecting viruses, release
     occurs when the cell fills with new virions and lyses, spilling the new viruses into the surrounding
     environment.  Some forms, however, take a much slower approach.  Enveloped viruses must
     wrap a bit of modified host-cell membrane around themselves, so release occurs by a viral
     enzyme-mediated process called budding, releasing just a few viruses at a time, until the host
     cell runs out of ATP and dies.

Bacteriophage Structure and Replication

     The T-even bacteriophage has a complex structure composed of a protein coat called, as previously stated a capsid, which is in turn composed of individual protein subunits called capsomeres.  The capsid surrounds nucleic acid, either DNA or RNA, and is bound to a hollow protein sheath which surrounds a hollow protenaceous tube, which has lysozyme attached to its distal portion.  The lysozyme is used to digest away a portion of the cell wall of a host cell.  The sheath is attached to a base, which has many protein tail fibers. These serve to attach the bacteriophage to the host cell.

     When a bacteriophage contacts a host cell, it attaches itself by protenaceous tail fibers, which change their conformation on cell contact, allowing a base plate to come into contact with the host cell wall.  Small protein spikes penetrate the wall (G+ host) or membrane (G- host) to hold the virus in place, then the sheath contracts, bringing the tube into contact with the cell.  Lysozyme digests a small hole at the point of contact, so the tube can penetrate between the cell wall and the inner cell membrane.  The viral nucleic acid is then injected into this space, where it is transported passively into the cytoplasm of the host.  If the bacteriophage injects DNA, this serves as a template for mRNA, which codes for the production of early proteins.  These early proteins include viral structural components and enzymes which enable the replication of new viral DNA, and lysozyme.  The new virions begin to assemble or amplify within the host cell, until there is no more room in the cytoplasm.  Lysozyme then causes lysis of the cell wall, and the new virions are freed to infect new host cells.

     Some bacteriophages contain RNA which is designated as either + or - stranded.  + (plus) stranded RNA serves immediately as mRNA, so that early viral proteins can be produced within the host cell cytoplasm. - (minus) stranded RNA serves as a template to produce new + stranded RNA, which then can be used to produce early proteins.  Other bacteriophages contain double-stranded RNA, which when in the cytoplasm of the host cell separates into + and - strands.  In all of the RNA bacteriophage types, assembly and release of new virions occurs in a fashion which is similar to that of the T-even DNA bacteriophages, by lysis of the host cell.

The Bacteriophage Replication Curve
     Since all of the replicative events which lead to the amplification of new virions occurs within a single bacterial host cell, the growth curve of the bacteriophage is essentially a one-step process, composed of: (1) an eclipse phase, encompassing penetration, replication of the viral nucleic acid, and the production of early proteins, followed by (2) a latent phase, wherein assembly of late proteins and replicated nucleic acid into new virions occurs, and finally (3) release of the new virons by lysis.  When this process is plotted as increase in viral numbers over time, it produces an S-shaped curve.  The number of new virions produced within the host cell varies greatly between different bacteriophage types (T-even bacteriophages number about 200 before lysis occurs) and represents the burst size for the amplified population.
Lysogeny and Lysogenic Conversion
      Viral infection does not always result in amplification and lysis.  If viral DNA incorporates itself as a prophage within the host chromosome, the cell may not immediately enter into the lytic cycle, or even enter the lytic cycle at all.  This phenomenon is called lysogeny, or the temperate phase of the viral cycle.  A prophage-infected bacterial cell cannot be re-infected by a virus of the same type, and may continue to survive with no ill effect at all, even replicating and passing its recombined genome on to new generations of progeny cells via binary fission.  All of the progeny cells could then be induced to enter into the lytic cycle, or may continue to survive unaffected by the replicated viral DNA in their chromosomes.  The inactivity of prophage genes arises from the presence of a regulatory gene already present in the host cell, which inhibits their transcription unless it has been damaged by environmental stress, such as exposure to ultraviolet radiation.

      In some instances, however, phenotypic properties of a prophage-infected host are altered owing to the presence of viral DNA.  This phenomenon is called lysogenic conversion, and can result in the production of structures or substances which enhance the pathogenicity of some bacteria.  Examples of species which become pathogenic via lysogenic conversion include Corynebacterium diptheriae, the agent of diptheria, Clostridium botulinum, which causes botulism, Staphylococcus aureus, an inhabitant of the nasal cavity which causes food intoxication, toxic shock syndrome, scalded skin syndrome in infants, as well as boils and other forms of localized inflammations, and Streptococcus pyogenes, the agent of strep-throat, scarlet fever, and necrotizing faciatus.  Each of these organisms produces exotoxins only when infected by a prophage.

     The process of viral infection of a host cell including the incorporation of viral DNA into the host cell chromosome is called transduction.  In generalized transduction, the cell either enters into the lytic cycle and lyses, releasing new virions into the surrounding environment, or the viral DNA becomes a temperate prophage.  In this instance, only viral DNA recombines with that of the host cell.  Occasionally, however, some of the host cell DNA remains incorporated with that of the replicating viral genome during the assembly phase of the lytic cycle.  Any new virion containing this DNA is said to be a defective phage, since it will be incapable of pre-empting the normal metabolism of the host cell it infects.  When the genome of a defective phage incorporates with that a host cell, the process is called specialized transduction, and can allow the host to express phenotypic characteristics it normally would not have through lysogenic conversion.
Viral Replication in Plant Cells
     It is difficult for viruses to invade plant tissue, since the outer epidermis of most plants is either covered by a layer of waxy cuticle, or by layers of cork cells which make up bark.  Therefore, most viral infections can only occur if there is injury to the outermost layer of tissue, either by abrasion or insect bite.  When a plant virus does gain entry into the inner plant tissue, the entire virus enters the host cell cytoplasm and uncoating occurs there.  Plant virus replication always results in cell lysis.
One example of a plant virus is the tobacco mosaic virus (TMV).  It was the first virus to be identified in 1898 by Martinus Beijerinck. This virus is rod-shaped and contains RNA.  It generally gains entry to plant tissue via abrasion, then replicates within the host cells, causing them to lyse.  The term "mosaic" refers to the patchy yellow chlorotic spots on the leaves of the infected plant leaves which indicate that the virus has killed photosynthetic tissue.
Viral Replication in Animal Cells
     Viruses which infect animal cells may contain either DNA or RNA, and may or may not be surrounded by an envelope.  While some animal viruses are uncoated outside of the host cell (adenoviruses and polioviruses bind to receptor sites on the cell membrane, then flatten out and "push" their nucleic acids in the host cell cytoplasm), and others, such as the herpesvirus are uncoated directly in the cytoplasm, most enter through endocytosis.  In this process, the virus is surrounded by the host cell plasma membrane.  The newly- formed vesicle (vacuole) is then bound to a lysosome, which releases lysozyme into the vacuole (phagolysosome).  Lysozyme digests the capsid, releasing viral nucleic into the phagolysosome.  The nucleic acid then passes through the vacuolar membrane, into the host cell cytoplasm.

    The synthesis of early and late proteins, as well as the synthesis of new nucleic acids dependes on the nature of the type of animal virus nucleic acid.  Viruses which contain double-stranded DNA take several hours to uncoat, then enter the host nucleus and stop normal biosynthetic activities.  These viruses serve as templates for their own replication, and are released by lysis.  Single-stranded DNA viruses uncoat, then utilize host cell components to synthesize their missing complementary DNA nucleoside.  After the double strand is produced, it can serve as a template for replication.  Release is also by host cell lysis.  RNA viruses can also be double or single stranded.  Double-stranded RNA viruses do not enter the nucleus of the host cell; they contain their own RNA polymerase which is used for self-replication of viral nucleic acids upon uncoating in the host cell cytoplasm.  Once the two pieces of RNA have been separated, the + strand serves as mRNA to build new early proteins.  These are assembled into capsids surrounding the plus strand and integral RNA replicase then is used to synthesize - strands.  The new virions are released by host cell lysis.  Single-stranded RNA viruses contain either + or - strands.  The poliovirus, which is a + stranded form, uses its RNA as mRNA to produce a large polypeptide which is enzymatically cleaved to produce RNA polymerase.  This is then used to synthesize - RNA strands and early capsid proteins.  The assembly of new virions triggers the breakdown of host lysosomes, leading to host cell autolysis. This releases the newly formed viruses into the interstitial fluids, where they can infect new cells.  The influenza virus, a - stranded form, replicates after uncoating by using its RNA as a template to make new + strands, which serve to produce early proteins.  Some of these early proteins serve as capsid components, while others, neuriminidase and  hemagglutinin, are incorporated into the cell membrane of the host.  The new virions are releasd by budding, meaning that a portion of the host cell membrane surrounds each new capsid as it is released from the cell.  This membrane becomes the viral envelope, which enables the new virus to mimic the antigenic structure of its host.  The placement and composition of neuraminidase and hemagglutinin spikes on the envelope of an influenza virus is highly  variable, with as many as eight different types of - strands.  This is due to two phenomena called antigenic drift and antigenic shift.  Antigenic drift is caused by mutation in viral genes which code for the production of both of these spikes in newly replicated viruses.  Antigenic shift occurs due to gene reassortment when two different strains of an influenza virus infect a single host cell, leading to recombination of the viral genome prior to synthesis of early proteins and synthesis of new virions.  Drift and shift of viral antigens are important sources of variation in the influenza virus and can lead to the development of strains which are not as likely to be recognized and targeted by host immune responses, increasing pathogenicity.

     Retroviruses contain two strands of RNA, plus the enzyme reverse transcriptase.  These enveloped viruses attach themselves to a host cell by binding protein "spikes" to cell membrane receptor sites, such as the CD4 (Cluster of Differentiation) sites on the helper T4 cells of the cell mediated immune system in humans.  After uncoating in the host cell cytoplasm, the viral RNA acts as a template for the formation of  viral DNA strands, as reverse transcriptase binds DNA nucleotide bases to each RNA strand.  The new viral DNA enters the nucleus of the host cell and incorporates with host cell DNA.  Once incorporated, it can remain temperate for many years as a provirus, since host cells maintain regulatory genes which prevent the provirus from entering into the lytic cycle.  Eventually, however, due to environmental stress factors such as exposure to radiation or chemicals, the provirus will begin the process of transcription, and new viral early capsid and spike proteins, RNA, and reverse transcriptase.  Release of the new virions occurs by budding, which eventually kills the host cell due to loss of ATP.  Since retroviruses such as HIV (Human Immunodeficiency Virus) kill the helper T cells vital for the proper activation and control of the immune response, the host will ultimately die of secondary infections caused by opportunistic bacteria, fungi, protists, or other viruses.
     The incorporation of retroviral DNA as a provirus into the genome of a host cell can lead to the development of a latent infection, as in the case of HIV, but can also potentially alter the normal growth and development of a cell while not leading to the production of new virions.  The term oncogenesis (cancer formation) refers to the alteration of cell growth and speed of reproduction by a gene or genes.  These genes are called oncogenes, and are hypothesized to have been derived from part of the proviral genome, a phenomenon which is analogous to the lysogenic conversion of some bacterial cells.  While oncogenesis can also be triggered by exposure to carcinogenic chemicals and radiation, the pathway by which some cells become cancerous after exposure to certain viruses is through the transcription of viral messenger RNA which leads to the synthesis of kinase enzymes.  Viral oncogenic kinases strip phosphate from ATP and use it to phosphorylate the amino acid tyrosine.  Regulatory proteins which contain phosphorylated tyrosine trigger hyperplasia (increased cell reproduction) and hypertrophy (abnormal cell growth).  These abnormal cells overgrow normal healthy tissue, depriving it of nutrients and oxygen.  Examples of viruses may which carry oncogenes include the Epstein-Barr virus, the papilloma (wart) virus, HTLV1 and HTLV2.

Viral Classification

     Since viruses do not have all of the characteristics of living organisms, then way they are classified is considered separately from other biological forms.  The International Commission on Taxonomy of Viruses (ICTV) was founded to develop a taxonomic scheme.  This system of viral classification varies in that, while animal and bacterial virologists classify viruses on the basis of families, genera, and species in a very similar manner to the Linnean system used in other taxonomic schemes, plant virologists do not, tending instead to classify on the basis of shape, type of nucleic acid, mode of transmission, size, and other factors.  Approximately 1400 viruses have currently been identified, and these have been placed into 61 families.  Twenty-one of these families are composed of viruses which infect animal cells.  Animal viruses are generally placed into classes on the basis of the following characteristics:

1. Type of nucleic acid- DNA or RNA.
2. Type of nucleic acid- single or double stranded.
3. Capsid morphology- helical, bullet-shaped, isocohedral, circular, pleomorphic.
4. Presence or absence of an envelope.

There are five classes of RNA viruses, and three classes of DNA viruses which infect animal cells.

Cooperative Learning Activities

1.  Make a list of the characteristics of living things, then decide which of these characteristics
     viruses have and which of these viruses do not have.  Do viruses share enough characteristics of
     life to to be considered living things?  Why or why not?

2.  Suppose you were asked to develop a chemotheraputic compound to treat individuals
     suffering form viral infections.  How would you attack this problem?  Could there be more than
     one way to prevent a virus from infecting a host cell, or would you attempt to stop the virus
     from replicating once infection has taken place?  Defend your answer(s).

3.  In terms of the ability to infect animal cells, do enveloped viruses have any advantage(s)
     over nonenveloped viruses?  Why do you think plant viruses such as the tobacco mosaic virus
     (TMV) have not evolved an envelope?

  Test Yourself- Use this quiz to test your understanding of viruses.

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