Eukaryotic Cellbased Defenses Against Virus Replication Interferon

The clonal selection of antibody-producing B cells and effector T cells provides an exquisitely sensitive means for the infected host to specifically deal with invading microorganisms and viruses, and to eliminate virus-infected — and thus damaged — cells. However, it does take time for an effective defense to be mounted. There are more rapid if less specific defenses available locally. As briefly discussed in Chapter 7, these include the action of proteins with broad-spectrum antipathogen activities, inflammation, temperature rise, and interaction with nonspecific phagocytic cells of the immune system.

The ability of cells to produce interferon (IFN) provides another important rapid response. The cells capable of such a response contain a complex set of gene products that can be induced in direct response to virus attack and that render neighboring cells more resistant to virus replication. IFN has a large number of biological effects including the following:

  • Inhibition of virus replication in IFN-treated cells (target cells).
  • Inhibition of growth of target cells.
  • Activation of macrophage, natural killer cells, and cytotoxic T lymphocytes.
  • Induction of MHC-I and MHC-II antigens and Fc receptors.
  • Induction of fever.

A protein secreted from a cell in order to induce specific responses in other cells having specific receptors for it is generally termed a cytokine. IFN is one major group, but there are many others. For example, the proliferation of B cells responding to the presence of an antigen and helper T cells is the result of specific lymphocyte cytokines (an interleukin) secreted by the helper T cell.

It was shown in the late 1950s that culture media isolated from fibroblasts infected with certain viruses contained a substance or substances that would render uninfected cells more resistant to infection with similar viruses (i.e., the infected cells produced a substance that interfered with subsequent infection). Classic protein fractionation methods demonstrated that this substance — IFN — is actually a group of proteins, all very stable to acid pH and all able to function at very high dilutions, so that only a few molecules interacting with a target cell render that cell resistant to viral infection.

There are two basic interferons, types I and II. Type I IFNs are stable at acid pH and heat. All are distinct and are encoded by separate cellular genes, but all have the same general size and have roughly similar effects. The two major type I IFNs are IFN-a, expressed by leukocytes, and IFN-P, expressed by fibroblasts. There are at least three others in this class. There is only one type II IFN, IFN-y expressed mainly by T lymphocytes. Type I IFNs are most active against virus infections while IFN-y modulates the immune response, and appears to have some antitumor activity. All IFNs are very species specific; therefore, human IFN is active in human cells, mouse IFN in mouse cells, and so on.

The characterization of IFN followed by cloning and expressing IFN genes resulted in a lot of excitement concerning its potential use as an antiviral and anticancer drug. The promise has yet to be fully realized; it is now known that IFN proteins are very toxic to cells and methods for their efficient delivery to regions of the body where it would be therapeutic have yet to be perfected. Thus, although it is clear that the IFN response has a role in natural recovery from virus infection and disease, its complete therapeutic potential is yet to be fully exploited.

Induction of interferon

IFN induction takes place in the infected cell in response to viral products. A major inducer is double-stranded RNA (dsRNA), which is generated in infections by many RNA and DNA viruses. In addition, some viruses (e.g., reoviruses) use dsRNA as their genetic material. A single molecule of dsRNA can induce IFN in a cell under the appropriate conditions. A cellular protein, RIG-1 binds dsRNA to domains in its C-terminal region and serves as the cellular detector of such RNA. When bound with dsRNA, the RIG-1 protein activates a number of cellular transcription factors, which act together to induce expression of type I interferons. Interestingly, another antiviral protein expressed by mitochondria (MAVS - mitochondrial antiviral signaling protein) mediates the effect of RIG-1. The activation of a receptor protein by its binding to the appropriate signaling ligand leading to interaction and activation of further proteins leading to a transcriptional or other cellular response is a common feature of cellular signaling cascades, and will be discussed briefly in Part III, Chapter 13.

Because IFN is expressed from cellular genes, only cells that are relatively intact and functioning when dsRNA is present will express it. The requirement for continuing cell function is one reason why viruses that replicate slowly are good IFN inducers. When a virus capable of rapid replication and quick host-cell shutoff initiates an infection under optimal conditions, little IFN is generally induced.

The antiviral state

IFN inducers cause the cell in which they are present to synthesize IFN. This protein is secreted and interacts with neighboring cells to put them in an antiviral state in which antiviral effector molecules (AVEMs) are expressed. Cells that have been induced by IFN express new membrane-associated surface proteins, have altered glycosylation patterns, produce enzymes that are activated by dsRNA to degrade mRNA, and inhibit protein synthesis by ribosome modification. These effects are outlined in Fig. 8.1. In the antiviral state, thus, the cell is primed to trigger a number of responses to virus infection. As in the case of IFN induction, viral dsRNA acts as the trigger of these responses.

To date, expression of more than 300 cellular genes has been demonstrated to be induced or enhanced by IFN — many of these are involved in the establishment of the antiviral state. One — Mx — protein appears solely directed against influenza virus infections, although it also has activity against vesicular stomatitis virus (VSV). Some of these proteins that serve as antiviral effector molecules are listed in Table 8.2. Different mechanisms are involved in the different cellular responses to virus infection. Changes to the cell surface may make it more difficult for viruses to attach and penetrate. When presented with dsRNA, the antiviral cell activates 2',5'-oligoA synthetase — enzymatic activity that is induced by IFN, which produces an unusual oligonucleotide, 2',5'-oligoA. This, in turn, activates a latent mRNA endonuclease (RNAse-L). Finally, this endonuclease rapidly degrades all mRNA (viral and cellular) in the cell. The IFN-primed cell also expresses a dsRNA-dependent protein kinase (PKR) that causes modifications resulting in partial inactivation of the translational initiation factor eIF2 in the presence of dsRNA. This makes the cell a poor producer of virus proteins, and thus, an inefficient producer of new infectious virus, since all molecular processes are inhibited.

The action of IFN on cells is not always beneficial. Because IFN also acts as a negative growth regulator (the basis of its activity against tumor cells), its presence can interrupt the function of differentiated cells and tissues. Also, one cellular response to virus infection is the induction of a number of cellular genes that lead to programmed cell death (apoptosis); this process is outlined in more detail in Chapter 10, Part III. Such cell death is good for the host, since the reduction of virus replication is well worth the loss of a few cells, but in some cases IFN can block the induction of apoptosis and, thus, actually protect virus-infected cells! Further, IFN causes tissue inflammation and high fevers.

The toxic effects of the IFN response are alleviated by its being carefully balanced and controlled so that it is maintained only as long as needed. The amount of IFN produced by any given infected cell is very small, so that only the cells within the immediate vicinity are affected and converted to the antiviral state. If the cells are not infected, they may eventually recover and resume their normal processes.

Measurement of interferon activity

IFN activity is measured in a number of ways because there are so many different types and different effects. An easy and rapid method in virology is the plaque reduction assay. This method

  1. 8.1 The cascade of events leading to expression of interferon (IFN) and induction of the antiviral state in neighboring cells. The interferon inducer (dsRNA) produced during virus infection leads to an infected cell secreting small numbers of the IFN proteins, which are extremely stable glycoproteins. These interact with neighboring cells to induce the antiviral state in which a number of antiviral effector molecules (AVEMs) are expressed and can be triggered by the presence of dsRNA to alter the cell to markedly reduce the yield of infectious virus. dsRNA = double-stranded RNA; 2',5'-OAS=2',5'-oligoA synthetase; PKR=dsRNA-dependent protein kinase; tscp = transcription.
  2. 8.1 The cascade of events leading to expression of interferon (IFN) and induction of the antiviral state in neighboring cells. The interferon inducer (dsRNA) produced during virus infection leads to an infected cell secreting small numbers of the IFN proteins, which are extremely stable glycoproteins. These interact with neighboring cells to induce the antiviral state in which a number of antiviral effector molecules (AVEMs) are expressed and can be triggered by the presence of dsRNA to alter the cell to markedly reduce the yield of infectious virus. dsRNA = double-stranded RNA; 2',5'-OAS=2',5'-oligoA synthetase; PKR=dsRNA-dependent protein kinase; tscp = transcription.

is quite sensitive; it has been claimed that as few as 10 molecules of IFN can be detected with its careful use. Plaque assays are described in detail in Chapter 10, but in essence the process is as follows: Duplicate cell cultures are set up (see Chapter 9), and one culture is treated with IFN for several hours to allow the potential antiviral state to develop. Both are then infected

BASIC VIROLOGY PART II BASIC PROPERTIES OF VIRUSES AND VIRUS-CELL INTERACTION Table 8.2 Some antiviral proteins induced or activated by interferon.

Protein

Function

2',5'-Oligoadenylate synthetase dsRNA-dependent protein kinase (PKR)

RNAse-L

MHC-I

Activates latent RNAse-L Phosphorylates eIF2 mRNA degradation Transcriptional regulation Antigen presentation

Specific blockage of influenza (and vesicular stomatitis virus) entry with the same number of infectious units of indicator virus (often VSV since it is so sensitive to IFN). The IFN-treated cells will produce fewer and smaller centers of virus infection (plaques) than will the untreated control. Serial dilutions of the original sample can be made until the effect is no longer seen, and a measure such as median effective dose (ED50) can be calculated. The ED50 is that dilution in which the number of plaques is reduced by 50% or plaques are 50% smaller than untreated ones. This reduction can be related to units of IFN activity and to the number of IFN molecules present.

Other cellular defenses against viral infection

Small RNA-based defenses

Discoveries starting in the early 1990s have demonstrated that small RNA molecules with double-stranded regions have a number of important roles in regulating eukaryotic cellular processes and protecting against pathogens beyond the induction of interferons. This is briefly described in Chapter 13 (Part III); here it suffices to note that there are pathways in eukaryotic cells for processing small RNA molecules encoded in the cell's genome into 22 base pair (bp) double-stranded RNA molecules (microRNAs or miRNAs). These miRNAs then bind to specific viral or cellular mRNA molecules, leading to their degradation.

Cells have a similar way of dealing with double-stranded RNA occurring in transcripts, such as those produced in viral infections. These can be processed into 28 base pair double-stranded RNA molecules called small interfering RNAs (siRNAs). Such siRNAs interfere with the translation of mRNAs containing homologous sequences also by inducing the degradation of those mRNAs. Thus, infection of a plant cell with a virus will lead to the spread of these to neighboring and more distant cells resulting in resistance to viral spread. The presence of plant virus genes, which act to counter the function of plant siRNAs, demonstrates the extent of this system in the plant kingdom.

It is not yet clear just how extensive the roles of miRNA and siRNA are in protecting animal cells, but evidence of the importance of these molecules in virus infections and the antiviral response is growing. There are between 200 and 300 miRNA-encoding sequences in the human genome and recently miRNAs have been identified in a number of DNA viruses including human herpesviruses and SV40. While it is often difficult to identify the target of a given miRNA, an SV40 miRNA blocks part of cellular control of its replication cycle. On the other hand, it has also been found that hepatitis C virus utilizes a small RNA species of human liver cells to increase the efficiency of translation of its mRNA. All available evidence suggests that siRNAs and miRNAs act as a kind of innate immune response directed against viral RNA motifs. There is also evidence that viruses may have co-opted miRNAs that target cellular defense mechanisms as a means of evading host responses.

Enzymatic modification of viral genomes

Another form of cell-based antiviral activity can be seen in responses to retrovirus infection by mammalian cells. A group of cytidine deaminases termed APOBECs recognize newly synthesized retroviral DNA generated by reverse transcriptase and deaminate cytidines in that DNA to yield uracils. This leads to hypermutation and inactivation of the virus or to degradation of the altered DNA strand. This process is so effective that HIV has a specific viral gene directed against APOBEC activity!

ANTIVIRAL DRUGS

All drugs effective against pathogenic microorganisms must target some feature of the pathogen's replication in the host that can be efficiently inhibited without unduly harming the host. Some drugs are effective against the earliest stages of infection and can be given to an individual before he or she is exposed or for a short time after exposure. Such prophylactic use cannot be effective in large populations except under very specific circumstances (e.g., military personnel prior to entering a biological hazard zone).

Despite the value of some prophylactic drugs, the most desirable drugs are those that can effectively interrupt the disease at any stage. The dramatic effectiveness of penicillin in treating numerous bacterial infections after World War II has proved a model for such drugs, but the earliest specific antibacterial drugs were made up of complex organic molecules containing mercury that Ehrlich utilized to combat syphilis at the end of the last century. He termed these "magic bullets" and developed them to reduce the toxicity of mercury, whose use as an antisyphilitic agent was known to be effective since the Renaissance in Europe. Perhaps not surprisingly, Ehrlich's success was marred by the anger of some moralists who argued that the disease was a punishment for sin! While science progresses, society does so more slowly, and in the past few years similar arguments have been made against developing treatments for

AIDS.

The problem of therapeutic drug toxicity is a continuing one. Many effective inhibitors of metabolic processes, even if more or less specific for the pathogen, will have undesirable side effects in the person being dosed. The general ratio of benefit of a drug to its undesirable side effects is termed the therapeutic index. Determination of a drug's therapeutic index requires extensive animal testing and extensive documentation, and is a major factor in the expense involved in developing effective pharmaceuticals for any purpose.

Targeting antiviral drugs to specific features of the virus replication cycle

Given the fact that viruses are obligate intracellular parasites, it is easy to understand why a chemotherapeutic approach to halting or slowing a viral infection is difficult to achieve. Unlike bacterial cells, which are free-living, viruses utilize the host cell environment for much of their life cycle. Therefore, chemical agents that inhibit both virus and host functions are not a good choice for therapy.

The preferred strategy has been to identify the viral functions that differ significantly from or are not found within the host and are therefore unique. For each virus of clinical interest, a good deal of effort has been expended on understanding the virus's life cycle and attempting to develop drugs that can specifically block critical steps in this cycle. Table 8.3 lists targeted stages in the virus life cycle along with examples of existing or proposed agents that could block the cycle with some measure of specificity. With each of these, the problem of resistant mutants always arises, leading to limitation of the drug's usefulness.

Table 8.3 Some targets for antiviral drugs.

Step in virus life cycle targeted

Molecular target of inhibitor

Example

Virus attachment and entry

Surface protein-receptor interaction

Receptor analogues, fusion protein, amantadine

DNA virus genome replication

Viral DNA polymerase, thymidine

Acyclovir

kinase

RNA virus genome replication

Viral RNA replicase

Ribavirin

Retrovirus - reverse transcription

Reverse transcriptase

AZT, ddC, ddl

Retrovirus - integration

Integrase

In clinical trials

Viral transcriptional regulation

HIV tat

(Theoretical)

Viral mRNA posttranscriptional

HIV rev

(Theoretical)

processing (splicing)

Virion assembly

Viral protease

Protease inhibitors (ritonavir, saquinovir)

Virion assembly

Capsid protein-protein interactions,

Rimantadine, protease inhibitors

budding

Acyclovir and the herpesviruses

The development of acycloguanosine (acG) for use in herpesvirus infections marked a great advance in the chemotherapy of viral infections. This compound, prescribed under the name acyclovir, is the first of the nucleoside analogues that are chain-terminating inhibitors. The structure is shown in Fig. 8.2. When the triphosphorylated form of acycloguanosine is incorporated into a growing DNA chain in place of guanosine, no further elongation can take place because of the missing 3' OH.

The specificity of acyclovir for herpesvirus-infected cells results from two events. First, after the nucleoside is transported into the cell, it must be triphosphorylated to be utilized as a substrate for DNA replication. The first step in this process, the conversion of acG to the monophosphate (acGMP), requires the presence of the herpesvirus-encoded thymidine kinase (TK). Following this, a cellular enzyme is able to add the next two phosphates, producing the tri-phosphate acGTP. This acGTP inhibits the synthesis of viral genomes by acting as a substrate for herpesvirus DNA polymerase. When this happens, the DNA chain is terminated - no additional bases can be added because of the missing 3'-OH group. The drug will inhibit the viral enzyme about 10 times more efficiently than it will the cellular DNA polymerases.

As a result of the requirement for herpesvirus TK and the inhibition and chain termination of herpesvirus DNA synthesis, acyclovir is highly specific for herpes-infected cells and is non-toxic to uninfected cells. Acyclovir has been used successfully in both topical and internal applications with both HSV type 1 and HSV type 2. While both types of HSV readily mutate to resistance in the laboratory, in both cases the mutant viruses do not replicate well in humans, and cessation of drug treatment results in the rapid appearance of wild type virus with its accompanying drug sensitivity. This and the low toxicity of the drug have made acyclovir the most successful targeted antiviral drug yet produced.

Chemical modification of aG's structure has resulted in gancyclovir [9-(1,3-dihydroxy-2-propoxy)methylguanine] (Fig. 8.2). This drug has the same properties as acG, except that it is specific for cells infected with cytomegaloviruses. Unfortunately, this drug has a severe toxicity when given intravenously and must be used with caution.

Blocking influenza virus entry and virus maturation

Type A influenza viruses enter their host cells by means of the receptor-mediated endocytotic pathway. In this process, the viral hemagglutinin molecules in the membrane of the particles

OH H Arabinosyl cystosine
Fig. 8.2 The structure of some currently effective antiviral drugs.

undergo a conformational change when the pH of the endocytotic vesicle is lowered to around 5 after fusion of the vesicle with an acidic endosome. At this lower pH, the viral membrane undergoes fusion with the vesicle membrane and viral nucleocapsids enter the cell cytoplasm (see Chapter 6).

Two compounds that have been developed interfere with the ability of the cell to change pH within influenza A virus-modified vesicles — amantadine and rimantadine. Their structures are included in Fig. 8.2. Amantidine (1-aminoadamantane hydrochloride) is a basic primary amine, and can prevent the acidification that is essential for completion of viral entry.

The drug also works during virus assembly and maturation. At this time, newly synthesized hemagglutinin must be transported to the plasma membrane prior to particle budding. During this transport it is important that the exocytotic vesicle does not become acidified, or the hemagglutinin will assume its fusion conformation and be unavailable for correct assembly. The small viral protein M2 serves as an ion channel protein in the vesicle membrane that blocks this acidification. Thus, amantidine also inhibits the action of M2 and thus serves to block correct maturation of type A flu virus particles.

Amantidine must be administered as early as possible after the initial infection in order to have any efficacy in reducing disease symptoms. Prophylactic administration of the drug during epidemics is not considered to be a practical approach in the Western world because of the high dosages required and problems with side effects; it has been used with some success in isolated flu outbreaks in Russia, however. The related drug rimantidine appears to have fewer side effects and is now the preferred drug. Viral mutants resistant to both these drugs are readily observed; all have alterations in the M2 protein.

Since neuraminic acid on the surface of the host cell is a major receptor for influenza virus, drugs have been developed to serve as mimics in order to block the earliest steps in viral attachment and penetration. Two, Tamiflu and Relenza, are currently available. Of course, mutation to resistance will be a problem with these drugs also.

Chemotherapeutic approaches for HIV

When it was discovered that the viral agent that causes AIDS is, in fact, a retrovirus, the immediately obvious goal was the development of a drug that could specifically inhibit the unique viral replicative enzyme of the retroviruses: reverse transcriptase. A drug that had been developed as an antitumor agent was found to inhibit this enzyme: 3'-azido-2'3'-dideoxythymidine, commonly called azidothymidine or AZT (Fig. 8.2). Like acG, this drug, when transported into the cell and phosphorylated, can be utilized by the HIV polymerase to produce a chain termination because of the missing 3' OH. Although the drug exhibits a good specificity for HIV reverse transcriptase compared with cellular DNA polymerases in vitro, severe toxic effects are still seen when the drug is administered to patients. Most importantly, because of the high mutability of HIV replication (see Chapters 19 and 20, Part IV), the development of AZT-resistant mutants occurs rapidly.

Other nucleoside analogues have been produced for therapeutic use. Notable are dideoxy-cytidine (ddC) and dideoxyinosine (ddI). Since development of resistance to these two drugs does not occur in the mutation of the virus to AZT resistance, the drugs are commonly used in combination. Non-nucleoside analogue reverse transcriptase inhibitors have also been developed and approved for use in patients. These drugs are highly effective and less costly than other anti-HIV drugs, but viral resistance develops quickly so they can only be used for short periods alone or for longer periods in combination with other drugs (see below).

A major advance in the chemotherapeutic treatment of HIV infection was the production and use of the class of drugs known as protease inhibitors. Retroviruses, as well as many other viral families, require proteolytic processing of initial translation products so that the active viral proteins can be made. For HIV (like all retroviruses), this is carried out by a viral-encoded protease. The drugs act by inhibiting HIV protease; as a result, the posttranslational processing of viral products as well as the final proteolytic steps required during viral assembly are blocked (see Chapter 19).

The newest approved drug against HV is a viral entry inhibitor named Fuzeon. It is a peptide that has the same sequence as one of the alpha helical regions of the HIV envelope protein, gp4l. For the gp4l protein to function to promote viral-cell membrane fusion in HIV infection, this region must associate with another helical region in the same protein, and the presence of Fuzeon inhibits this pairing.

Multiple drug therapies to reduce or eliminate mutation to drug resistance

The most promising therapy against HIV now being used involves the use of multiple drugs. The original protocol required the simultaneous administration of AZT, another nucleoside analogue such as ddC, and a protease inhibitor. Initial results with this cocktail were quite impressive. Clinical observations of AIDS patients showed reversal of symptoms and rebound of levels of CD4 cells. Viral loads decrease and circulating virus all but disappears. With the wide application of these therapies in the United States, most cities reported a decrease in deaths from AIDS by the end of 1997. This therapy is called highly active antiretro viral therapy

(HAART) and entails the use of four inhibitors. For instance, one treatment uses a protease inhibitor (lopinavir) along with three reverse transcriptase inhibitors (3TC, tenofovir, and efa-virenz). Combinations of several drugs into single doses and other combinations are all available for use; in all of these cases rapid reduction in viral load is the objective.

This exciting picture must be tempered by words of caution. First, these therapies are quite complicated and expensive. They cannot be readily applied to developing nations and to individuals at risk who do not have the financial or emotional resources required for the treatment, which requires a lot of self-discipline. If dosages are skipped or missed, there is the great danger of developing resistant mutants that would effectively destroy progress made by the patient. This fear was recently underlined by the finding that even after long periods of treatment, HIV genomes still exist in critical lymphocytes and can be recovered as infectious virus if drug is removed. At this point, it is assumed that the therapy must be followed for the rest of the patient's life. There are no data yet on the long-term effects of this therapy. Thus, a major question yet looms: What will be the ultimate effect on the patient?

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  • bryan
    How interferons render cells resistant viruses?
    3 years ago

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