Viruses

Basic Properties and structure

Viruses are obligate pathogens in that they can only replicate inside the cells of the human body. The fact viruses use the host cell machinery for vital functions like replication means that it is very difficult to develop drugs which take advantage of differences between viruses and the host. For example, viruses uses the ribosomes of their host to replicate and thus drugs like antibiotics which work well with bacteria would not work well with viruses.

Viruses come in a variety of different sizes, morphologies and types of genomes such as the following.

• single stranded linear DNA (parvovirus) or RNA (like poliovirus)

• double-stranded linear DNA (like herpes viruses) or RNA (reovirus)

• circular single stranded DNA or double stranded circular DNA (polyoma viruses)

• double-stranded DNA with covalently linked proteins at the end of the DNA strands (adenovirus) or with each end covalently sealed (poxvirus)

• segmented where the virion contains multiple RNA genomic fragments (influenza) or non-segmented where the virion contains a single genome (HIV)

• polarity of genome in that the RNA can be sense (+) stranded or antisense (-) stranded. This will be important later in in respect to replication of the virus.

• nonenveloped or having an envelope surrounding a capsid which surrounds the genome.

3 types of proteins are common to all retroviruses (like HIV): (1) GAG proteins for the capsid, (2) Env proteins for the envelope and (3) Pol proteins for reverse transcriptase and integrase.

Growth of virus under benign laboratory conditions lacks the selective pressures of the body and allows weaker strains to survive. This process is used to develop attenuated virus strains for use in vaccines.

How Viruses Replicate

Replication depends on replication of the viral genome as well as the production of viral proteins which are assembled into progeny virions.

• DNA viruses will go to the nucleus (except poxviruses) where they use the cellular RNA polymerase to produce mRNA. Their genome is replicated by either host or viral DNA polymerase.
• RNA of positive strand RNA viruses is a mRNA (that is infectious) that can be translated. The RNA of positive strand RNA viruses is an mRNA (except retroviruses). Their genome is replicated by a viral RNA dependent RNA polymerase (transcriptase) in the cytoplasm.
• Negative strand RNA viruses carry in a virion transcriptase (RNA-dependent RNA polymerase) to produce a positive complementary mRNA as well as to replicate their genome in the cytoplasm.
• Retroviruses convert their RNA to DNA by a virion reverse transcriptase. DNA integrates into the host nuclear DNA followed by production of mRNA by the host RNA polymerase. Such viral reverse transcriptase that produces DNA from the viral RNA cannot correct nucleotide misincorporation errors. As a result, retroviruses acquire on average one point mutation every replication cycle. A typical untreated HIV infection may eventually involve HIV genomes with every possible point mutation. This explains why the virus can switch from infecting macrophages to infecting T cells and can quickly acquire resistance to drugs. The error rate, however, is not so high as to accumulate so rapidly that the virus can not survive. (rapid viral diversification in one host does not necessarily lead to rapid evolution of the virus in the population as a mutated virus may not be able to infect a new host). DNA integrates into the host DNA followed by production of mRNA by the host RNA polymerase.

Steps of Virus Replication

1) Entry and Uncoating: specific proteins of the virion display attachment proteins. The host cell may display surface cellular receptors composed of glycoproteins or polysaccharides. Viral attachment proteins (VAPs) that bind to erythrocytes are termed hemagglutinins.

Adsorption is mediated by high affinity interaction between the viral attachment proteins and receptors.

Viruses that infect animal cells typically use cell surface receptor molecules on the host cell that are either very abundant (such as sialic-acid containing oligosaccharides in the case of influenza) or those which are uniquely found on those types in which the virus can replicate.

Often a single type of receptor is used by many types of viruses and some viruses can use several different receptors. Different viruses that infect the same cell type may even use a different receptor. For example, hepatitis which is caused by at least 6 viruses all preferentially replicate in liver cells. Receptors for 4 of the hepatitis viruses are all different. Receptors do not need to be proteins, the herpes simplex virus, for example, binds to heparan sulfate proteoglycans through specific viral membrane proteins. Frequently, viruses require both a primary receptor and a secondary co-receptor for effective attachment and entry into host cells as is the case with HIV.

Viruses that bind to receptors expressed on specific cell types may be restricted to certain species (host range) like human or mouse or specific cell types. The susceptible target cell defines the tissue tropism (e.g., neurotropic, lymphtropic). Chemokine receptors are often used such as the B-chemokine receptor (CCR5) and alpha-chemokine receptor (CXCR4) which are used by HIV.

Most nonenveloped viruses enter the cell by receptor-mediated endocytosis or by viropexis (direct penetration of the membrane). Endocytosis is a normal process used by the cell for uptake of receptor-bound molecules such as hormones and low density lipoproteins.

Enveloped virus enter the host cell by fusing either with the plasma membrane (e.g., HIV) or with the endosomal membrane following endocytosis (e.g., influenza virus).. Fusion is thought to be similar to a SNARE mediated fusion of vesicles.

Once into the cell, DNA viruses go to the nucleus (except for poxviruses). RNA viruses remain in the cytoplasm (except for retroviruses).

2) Production of early mRNA and non-structural proteins

3) Production of viral genome

4) production of late mRNA and structural proteins

5) assembly and release: The site and mechanism of virion assembly in the cell depends on the location of genome replication and whether the final structure is a naked capsid or enveloped virus. The assembly process begins when the necessary pieces are synthesized and the concentration of structural proteins in the cell is sufficient to thermodynamically drive the process, much like a crystallization reaction.

• cubical viruses: first a pro-capsid forms and then nucleic acid is spooled into the capsid (bacteriophages use this mechanism of assembly).
• retrovirus structural proteins accumulate at the virus glycoprotein saturated cell membrane and form the virion during "budding".
• release of new virus by budding or exocytosis: Here, the nucleocapsid takes up a coat of the cell membrane while exiting the cell. This occurs with enveloped viruses. For example, the herpes simplex virus nucleocapsid assembles in the nucleus and buds at the nuclear membrane.
• naked capsid viruses are generally released after lysis of the cell.

How Viruses Cause Disease

A single virus particle (virion) that infects a single host cell can produce thousands of progeny in the infected cell. The cell often breaks open (lyses) and thereby allows the progeny viruses access to nearby cells. Many of the clinical manifestations of viral infection reflect this cytolytic effect. For example, the cold sores formed by herpes simplex virus and the lesions caused by the smallpox virus reflect the killing of the epidermal cells in the local area of infected skin.

About 15% of all malignant cancers are also cause by viruses. Hepatitus B virus, for example, is associated with liver carcinoma. Certain types of papillomarviruses are associated with cervical and penile cancer. These types of papillomaviruses exist as an episomal viral DNA and integrate into the host DNA thereby disrupting the negative regulator E2 resulting in strong activation of E6 and E7 oncogenes that bind and inactivate p53 and Rb.

Just as the genotype of the invading virus can influence the course of a viral infection, so too can the genetic background of the invaded host. For example, mousepox is a generalized infection caused by the ectroemlia virus, whihc is an inapparent disease in the genetically reistanct C57B1/6 mouse strain. On the other hand, only one infectious particle of the same virus can result in 100% mortality in the sensitive BALB/c or A mouse strains. This difference in strain susceptibility is related to differences in the cytokine profile produced after infection. In the C57B1/6 strain, there is a rapid induction of type 1 cytokiens and a potent cytotoxic T lymphocyte resposne, whereas in the BALB/c or A mice these cytokines are virtually absent with a complete absence or delayed induction of a CTL response.

How are Viruses Detected by the Immune System?

Virus Identification and Detection

Some ways to identify and detect viruses include the following:

• PCR where nucleic acid is extracted from a specimen (if RNA virus, RNA is converted to cDNA by reverse transcriptase) viral DNA is amplified by virus-specific primer pairs and DNA fragment is analyzed by agarose gel electrophoresis.
• ELISA where viral antigen is bound to plastic surface and antibodies in the patient's sera specifically bind the viral antigen. This antigen-antibody complex is then reacted with anti-human IgG conjugated with an enzyme (e.g., alkaline phosphatase). This complex is then visualized by adding chemical colorigenic substrates.
• Western Blot is a more confirmatory assay than ELISA for HIV. Viral proteins (e.g., gp120, gp41 and p24 in the case of HIV) are transfered to filter and filter is incubated with patient's serum. Filter is then incubated with labeled anti-human Ig serum.

Treatment

Despite the fact that viruses use the machinery of the host, their method of operation can be different. Most antiviral drugs are nucleoside analogues which inhibit viral polymerases. Many viral polymerases are less specific for substrate than are host enzymes. The viral polymerase will bind a nucleotide analogue with modifications of the base and/or sugar much better than the host enzyme. These drugs prevent chain elongation or proper recognition and base pairing.

Resistence to drugs can come about by selective pressures that lead to the emergence of mutant viruses. These selective pressures include (1) the immune response, (2) antiviral drugs and (3) opportunity to survive in new species (emerging infections). Mutations are more frequent in viruses due to the lack of proofreading function of their viral RNA and DNA polymerases. Some mutations that occur include the following:

• point mutations which typically lead to a gradual alteration of antigenic eptiopes or targets of antiviral drugs without loss of the primary function of the gene.
• deletions are observed only in non-essential viral genes (e.g., strains which lack thymidine kinase).
• homologous recombination occurs between genomes of genetically similar viruses. (e.g., HIV and SIV can recombine)
• genomic reassortment takes place when two segmented viruses infect the same cell and is unique to viruses.

Drugs which target the following processes have been effectively used against viruses.

• Non specific: interferons Virus infection and/or dsRNA induces IFN production and secretion. IFN binds to its receptor and induces STAT transcription factors leading to inactivation of elongation factor, RNAse induction and inhibition of cell proliferation.
• Uncoating: Uptake of many viruses utilize the acidic environment of the endocytic vesicle to initiate uncoating. Drugs like amantidine (which is effective against influenza A) can neutralize these compartments and inhibit virion uncoating.
• Genome replication: Antiviral drugs that cause termination of the DNA chain due to modified nucleoiside sugar residues include Acyclovir (used against herpes viruses that encode a thymidine kinase; the kinase activates the drug by phosphorylation. No initial phosphrylation occurs in uninfected cells)  and ganciclovir (used against CMV).  dideoxyinosine (DDI) and AZT are dideoxynucleotides that inhibit reverse transcription. Newer non-nucleotide non-substrate analogs like nevirapine bind and inhibit the HIV RT.
• mRNA synthesis: Ribavirin is an analog of the nucleoside guanosine and inhibits nucleoside biosynthesis and mRNA capping.
• Protein processing: Protease inhibitors like Saquinavir have been effective against HIV which needs a protease to chop up long transcripts.