*3.2.3. Transcription*

Once a virus genome becomes operational in a host cell, it then generates messenger RNA (mRNA) molecules that direct the synthesis of viral proteins. Production of mRNA is initiat‐ ed by proteins known as transcription factors. Several antivirals are now being designed to block attachment of transcription factors to viral DNA. Kao et al. recently identified a com‐ pound called nucleozin via random screening, which was found to inhibit influenza by in‐ teracting with influenza NP. Nucleozin causes the NPs to aggregate abnormally, and consequently inhibits normal viral transcription, crippling the replication cycle by extension [11]. Examination of a nucleozin analogue revealed that the compound functions by binding to two copies of NP and forming abnormal dimers, causing the proteins to aggregate and preventing them from functioning normally. Nucleozin was also shown to inhibit influenza virus in vitro and in a mouse model, making it a promising candidate for a new antiviral drug.

## *3.2.4. Translation/antisense*

Genomics has not only helped find targets for many antivirals, it has provided the basis for an entirely new type of drug, based on "antisense" molecules. These are segments of DNA or RNA that are designed as complementary molecule to critical sections of viral genomes, and the binding of these antisense segments to these target sections blocks the operation of those genomes. A phosphorothioate antisense drug named fomivirsen has been introduced, used to treat opportunistic eye infections in AIDS patients caused by cytomegalovirus, and other antisense antivirals are in development.

#### *3.2.5. Translation/ribozymes*

**Figure 3.** Example of the mechanisms of antivirals: Mechanism of action of azidothymidine (AZT). AZT needs to be phosphorylated, in three steps, to the triphospate form before it can interfere with the reverse transcriptase reaction

244 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Another target is integrase, which splices the synthesized DNA into the host cell genome. There appears to be no functional equivalent of the enzyme in human cells. The biochemical mechanism of integration of HIV DNA into the host cell genome involves a carefully de‐ fined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reac‐ tions [10]. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. However there are several promising candidates in several classes of compounds, including nucleotides, dinucleotides, oligonu‐ cleotides and miscellaneous small molecules such as heterocyclic systems, natural products,

diketo acids and sulfones, that have been discovered as inhibitors of HIV integrase.

*3.2.2. Integrase*

Yet another antiviral technique inspired by genomics is a set of drugs based on ribozymes, which are enzymes that will cut apart viral RNA or DNA at selected sites. In their natural course, ribozymes are used as part of the viral manufacturing sequence, but these synthetic ribozymes are designed to cut RNA and DNA at sites that will disable them.

A ribozyme antiviral to deal with hepatitis C has been suggested, and ribozyme antivirals are being developed to deal with HIV. An interesting variation of this idea is the use of ge‐ netically modified cells that can produce custom-tailored ribozymes. This is part of a broad‐ er effort to create genetically modified cells that can be injected into a host to attack pathogens by generating specialized proteins that block viral replication at various phases of the viral life cycle [12].

#### *3.2.6. Protease inhibitors*

Some viruses include an enzyme known as a protease that cuts viral protein chains apart so they can be assembled into their final configuration, such as Saquinavir (Figure 4). HIV in‐ cludes a protease, and so considerable research has been performed to find "protease inhibi‐ tors" to attack HIV at that phase of its life cycle. Protease inhibitors became available in the 1990s and have proven effective, though they can have unusual side effects, for example causing fat to build up in unusual places. Improved protease inhibitors are now in develop‐ ment [13].

As to the duration of treatment, this may vary from a few days (HSV, VZV, influenza virus infections) to several months or years (HIV, HBV and HCV infections), depending on whether we are dealing with an acute (primary (i.e. influenza) or recurrent (i.e. HSV, VZV) infection or chronic, persistent (i.e. HIV, HBV, HCV) infection. For HIV infections it is still being evaluated whether long-term treatment can be interrupted, without loss of benefit (or

Vaccines and Antiviral Agents http://dx.doi.org/10.5772/56866 247

While the short-term treatment (5–7 days) of HSV, VZV and influenza virus infections, and even the more prolonged treatment of CMV infections, can be based on single-drug therapy, for the long-term treatment of HIV infections combination of several drugs in a triple-drug cocktail (also referred to as HAART for 'highly active anti-retroviral therapy') has become the standard procedure, and likewise, the long-term treatment of HBV infections may in the

Pharmacokinetic parameters to be addressed, when evaluating the therapeutic potential, in‐ clude bioavailability (upon either topical, oral or parenteral administration), plasma protein binding affinity, distribution through the organism (penetration into the CNS, when this is needed), metabolism through the liver (i.e. cytochrome P-450 drug-metabolizing enzymes) and elimination through the kidney. Particularly when concocting the multiple-drug combi‐ nations for the treatment of HIV infection, possible drug–drug interactions should be taken into account: i.e. some compounds act as P-450 inhibitors and others as P-450 inducers, and this may greatly influence the plasma drug levels achieved, especially in the case of NNRTIs

Toxic side effects, both short and long-term, must be considered when the drugs have to be administered for a prolonged period, as in the treatment of HIV infections. These side effects may seriously compromise compliance (adherence to drug intake), and could, at least in

Finally, resistance development may be an important issue, again for those compounds that have to be taken for a prolonged period, as is generally the case for most of the NRTIs, NNRTIs and PIs currently used in the treatment of HIV infections. Yet, the nucleoside phos‐ phonate analogues (NtRTIs) tenofovir and adefovir do not readily or rapidly lead to resist‐ ance development, even after more than 1 year of therapy (for HIV and HBV, respectively). Resistance has been noted with HBV against lamivudine after long-term therapy (>6 months), but, if resistant to lamivudine, HBV infections remain amenable to treatment with adefovir dipivoxil. As has been occasionally observed in immunosuppressed patients, HSV may develop resistance to acyclovir, and CMV to ganciclovir, but, if based on ACV TK or CMV PK deficiency, these resistant viruses remain amenable to treatment with foscarnet and/or cidofovir [19]. In immunocompetent patients, treated for an acute or episodic HSV, VZV or influenza virus infection, short-term therapy is unlikely to engender any resistance

The evolution of viral vaccines from the time of Jennerian prophylaxis to today's recombi‐ nant technology has been a continuing story of success. From the relatively crude or "first generation" vaccines for smallpox, rabies, and yellow fever followed a second and third gen‐

part, be circumvented by a reduction of the pill burden to, ideally, once-daily dosing.

increased benefit) to the patient (structured treatment interruption, STI) [16].

future also evolve from single- to dual- or triple-drug therapy [17].

and PIs [18].

problems

**Figure 4.** Protease inhibitor antiviral Saquinavir.

*Structure: cis-N-tert*-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-2-quinolylcarbonyll-asparaginyl]-amino]butyl]-(4aS-8aS)-isoquinoline-3(S)-carboxamide methane sulfonate, hard gel capsules, Invirase®, also available as soft gelatin capsules (Fortovase®).

*Activity spectrum*: HIV (types 1 and 2).

*Mechanism of action*: transition-state, hydroxyethylene-based, peptidomimetic inhibitor of HIV protease.

#### **3.3. Release phase**

The final stage in the life cycle of a virus is the release of completed viruses from the host cell, and this step has also been targeted by antiviral drug developers. Two drugs named zanamivir (Relenza) and oseltamivir (Tamiflu) that have been recently introduced to treat influenza prevent the release of viral particles by blocking a molecule named neuraminidase that is found on the surface of flu viruses, and also seems to be constant across a wide range of flu strains [14].

#### **3.4. Considerations in the clinical development of antiviral agents**

A total of 37 antiviral compounds (not including interferons or immunoglobulins) have mo‐ mentarily been licensed for the treatment of HIV, HBV, herpesvirus, influenza virus and/or HCV infections [15]. In the preceding sections these compounds have been discussed from the following viewpoints: chemical structure, activity spectrum, mechanism of action, prin‐ cipal clinical indication(s). Other points that need to be considered before the full clinical po‐ tential of any given drug could be appreciated, are: (i) duration of treatment, (ii) singleversus multiple-drug therapy, (iii) pharmacokinetics, (iv) drug interactions, (v) toxic side effects and (vi) development of resistance. A particular issue that may be important in the clinical setting is whether the listed anti-HIV agents would be equally suited for the treat‐ ment of HIV-2 and HIV-1 infections.

As to the duration of treatment, this may vary from a few days (HSV, VZV, influenza virus infections) to several months or years (HIV, HBV and HCV infections), depending on whether we are dealing with an acute (primary (i.e. influenza) or recurrent (i.e. HSV, VZV) infection or chronic, persistent (i.e. HIV, HBV, HCV) infection. For HIV infections it is still being evaluated whether long-term treatment can be interrupted, without loss of benefit (or increased benefit) to the patient (structured treatment interruption, STI) [16].

causing fat to build up in unusual places. Improved protease inhibitors are now in develop‐

246 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

*Structure: cis-N-tert*-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-2-quinolylcarbonyll-asparaginyl]-amino]butyl]-(4aS-8aS)-isoquinoline-3(S)-carboxamide methane sulfonate,

*Mechanism of action*: transition-state, hydroxyethylene-based, peptidomimetic inhibitor of

The final stage in the life cycle of a virus is the release of completed viruses from the host cell, and this step has also been targeted by antiviral drug developers. Two drugs named zanamivir (Relenza) and oseltamivir (Tamiflu) that have been recently introduced to treat influenza prevent the release of viral particles by blocking a molecule named neuraminidase that is found on the surface of flu viruses, and also seems to be constant across a wide range

A total of 37 antiviral compounds (not including interferons or immunoglobulins) have mo‐ mentarily been licensed for the treatment of HIV, HBV, herpesvirus, influenza virus and/or HCV infections [15]. In the preceding sections these compounds have been discussed from the following viewpoints: chemical structure, activity spectrum, mechanism of action, prin‐ cipal clinical indication(s). Other points that need to be considered before the full clinical po‐ tential of any given drug could be appreciated, are: (i) duration of treatment, (ii) singleversus multiple-drug therapy, (iii) pharmacokinetics, (iv) drug interactions, (v) toxic side effects and (vi) development of resistance. A particular issue that may be important in the clinical setting is whether the listed anti-HIV agents would be equally suited for the treat‐

hard gel capsules, Invirase®, also available as soft gelatin capsules (Fortovase®).

**3.4. Considerations in the clinical development of antiviral agents**

ment [13].

**Figure 4.** Protease inhibitor antiviral Saquinavir.

*Activity spectrum*: HIV (types 1 and 2).

ment of HIV-2 and HIV-1 infections.

HIV protease.

**3.3. Release phase**

of flu strains [14].

While the short-term treatment (5–7 days) of HSV, VZV and influenza virus infections, and even the more prolonged treatment of CMV infections, can be based on single-drug therapy, for the long-term treatment of HIV infections combination of several drugs in a triple-drug cocktail (also referred to as HAART for 'highly active anti-retroviral therapy') has become the standard procedure, and likewise, the long-term treatment of HBV infections may in the future also evolve from single- to dual- or triple-drug therapy [17].

Pharmacokinetic parameters to be addressed, when evaluating the therapeutic potential, in‐ clude bioavailability (upon either topical, oral or parenteral administration), plasma protein binding affinity, distribution through the organism (penetration into the CNS, when this is needed), metabolism through the liver (i.e. cytochrome P-450 drug-metabolizing enzymes) and elimination through the kidney. Particularly when concocting the multiple-drug combi‐ nations for the treatment of HIV infection, possible drug–drug interactions should be taken into account: i.e. some compounds act as P-450 inhibitors and others as P-450 inducers, and this may greatly influence the plasma drug levels achieved, especially in the case of NNRTIs and PIs [18].

Toxic side effects, both short and long-term, must be considered when the drugs have to be administered for a prolonged period, as in the treatment of HIV infections. These side effects may seriously compromise compliance (adherence to drug intake), and could, at least in part, be circumvented by a reduction of the pill burden to, ideally, once-daily dosing.

Finally, resistance development may be an important issue, again for those compounds that have to be taken for a prolonged period, as is generally the case for most of the NRTIs, NNRTIs and PIs currently used in the treatment of HIV infections. Yet, the nucleoside phos‐ phonate analogues (NtRTIs) tenofovir and adefovir do not readily or rapidly lead to resist‐ ance development, even after more than 1 year of therapy (for HIV and HBV, respectively). Resistance has been noted with HBV against lamivudine after long-term therapy (>6 months), but, if resistant to lamivudine, HBV infections remain amenable to treatment with adefovir dipivoxil. As has been occasionally observed in immunosuppressed patients, HSV may develop resistance to acyclovir, and CMV to ganciclovir, but, if based on ACV TK or CMV PK deficiency, these resistant viruses remain amenable to treatment with foscarnet and/or cidofovir [19]. In immunocompetent patients, treated for an acute or episodic HSV, VZV or influenza virus infection, short-term therapy is unlikely to engender any resistance problems

The evolution of viral vaccines from the time of Jennerian prophylaxis to today's recombi‐ nant technology has been a continuing story of success. From the relatively crude or "first generation" vaccines for smallpox, rabies, and yellow fever followed a second and third gen‐ eration of improved or new viral vaccines. The application of techniques for attenuating, in‐ activating, and partially purifying candidate viruses yielded safe, effective vaccines against influenza, poliomyelitis, measles, mumps, and rubella. With the advent of effective national immunization programs in the United States and other areas of the world to promote wide scale use of these vaccines, we have seen a dramatic decrease in incidence of the viral infec‐ tions of children. The new biotechnology serves as the cornerstone for a fourth generation of vaccines and has already provided a licensed recombinant yeast human hepatitis B vaccine. The prospects for a wide spectrum of new or improved vaccines are highly encouraging, not only because of the recent technical advances but also because vaccine development has been recognized as a priority area of research. Under the National Institute of Allergy and Infectious Diseases' Program for Accelerated Development of New Vaccines, support is be‐ ing provided for developmental vaccine studies with hepatitis A and B, influenza A and B, rabies, rotavirus, varicella, and respiratory syncytial virus. The outlook for antivirals is equally optimistic. The same technologies that have provided greater insight into the genet‐ ics and molecular biology of viruses and hence the means to fashion subunit or even syn‐ thetic vaccines have yielded data that can be applied to successful development of targeted antiviral compounds.

[4] Tacket CO, et al. (1999) Phase 1 safety and immune response studies of a DNA vac‐ cine encoding hepatitis B surface antigen delivered by a gene delivery device. Vac‐

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[5] Roose K, Fiers W, & Saelens X (2009) Pandemic preparedness: toward a universal in‐

[6] De Clercq E (2004) Antiviral drugs in current clinical use. Journal of clinical virolo‐ gy : the official publication of the Pan American Society for Clinical Virology 30[2]:

[7] Teissier E, Penin F, & Pecheur EI (2011) Targeting cell entry of enveloped viruses as

[8] Doms RW & Moore JP (2000) HIV-1 membrane fusion: targets of opportunity. The

[9] Hay AJ, Wolstenholme AJ, Skehel JJ, & Smith MH (1985) The molecular basis of the specific anti-influenza action of amantadine. The EMBO journal 4[11]:3021-3024.

[10] Mathe C & Nair V (1999) Potential inhibitors of HIV integrase. Nucleosides & nucleo‐

[11] Kao RY, et al. (2010) Identification of influenza A nucleoprotein as an antiviral target.

[12] Olmstead AD, Knecht W, Lazarov I, Dixit SB, & Jean F (2012) Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target

for developing indirect-acting antiviral agents. PLoS pathogens 8[1]:e1002468.

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Shiffman ML (Springer, New York), pp 203-225.

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[13] Lamarre D, et al. (2003) An NS3 protease inhibitor with antiviral effects in humans

[14] Moscona A (2005) Neuraminidase inhibitors for influenza. The New England journal

[15] B. Kronenberger SZ (2012) Antiviral tagets in HCV. Chronic Hepatitis C Virus, ed

[16] Mancini E, Castiglione F, Bernaschi M, de Luca A, & Sloot PM (2012) HIV reservoirs and immune surveillance evasion cause the failure of structured treatment interrup‐

[17] Regidor DL, et al. (2011) Effect of highly active antiretroviral therapy on biomarkers

[18] Flentge CA, et al. (2009) Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs. Bioorganic & medicinal

of B-lymphocyte activation and inflammation. AIDS 25[3]:303-314.

fluenza vaccine. Drug news & perspectives 22[2]:80-92.

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tides 18(4-5):681-682.

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115-133.

#### **Author details**

#### Hongxuan He\*

Address all correspondence to: hehx@ioz.ac.cn

National Research Center for Wildlife Borne Diseases, Institute of Zoology, Chinese Acade‐ my of Sciences, Beijing, PR China

#### **References**


[4] Tacket CO, et al. (1999) Phase 1 safety and immune response studies of a DNA vac‐ cine encoding hepatitis B surface antigen delivered by a gene delivery device. Vac‐ cine 17[22]:2826-2829.

eration of improved or new viral vaccines. The application of techniques for attenuating, in‐ activating, and partially purifying candidate viruses yielded safe, effective vaccines against influenza, poliomyelitis, measles, mumps, and rubella. With the advent of effective national immunization programs in the United States and other areas of the world to promote wide scale use of these vaccines, we have seen a dramatic decrease in incidence of the viral infec‐ tions of children. The new biotechnology serves as the cornerstone for a fourth generation of vaccines and has already provided a licensed recombinant yeast human hepatitis B vaccine. The prospects for a wide spectrum of new or improved vaccines are highly encouraging, not only because of the recent technical advances but also because vaccine development has been recognized as a priority area of research. Under the National Institute of Allergy and Infectious Diseases' Program for Accelerated Development of New Vaccines, support is be‐ ing provided for developmental vaccine studies with hepatitis A and B, influenza A and B, rabies, rotavirus, varicella, and respiratory syncytial virus. The outlook for antivirals is equally optimistic. The same technologies that have provided greater insight into the genet‐ ics and molecular biology of viruses and hence the means to fashion subunit or even syn‐ thetic vaccines have yielded data that can be applied to successful development of targeted

248 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

National Research Center for Wildlife Borne Diseases, Institute of Zoology, Chinese Acade‐

[1] Steel J, et al. (2009) Live attenuated influenza viruses containing NS1 truncations as vaccine candidates against H5N1 highly pathogenic avian influenza. Journal of virol‐

[2] Kutzler MA & Weiner DB (2008) DNA vaccines: ready for prime time? Nature re‐

[3] Ramakrishna L, Anand KK, Mohankumar KM, & Ranga U (2004) Codon optimiza‐ tion of the tat antigen of human immunodeficiency virus type 1 generates strong im‐ mune responses in mice following genetic immunization. Journal of virology 78[17]:

antiviral compounds.

**Author details**

Address all correspondence to: hehx@ioz.ac.cn

my of Sciences, Beijing, PR China

ogy 83[4]:1742-1753.

9174-9189.

views. Genetics 9[10]:776-788.

Hongxuan He\*

**References**


[19] Bacon TH, Levin MJ, Leary JJ, Sarisky RT, & Sutton D (2003) Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clinical microbiology reviews 16[1]:114-128.

**Chapter 11**

**Viral Counter Defense X Antiviral Immunity in Plants:**

The history of plant virology dates to the late 19th century, when Iwanowski and Beijerinck, who were investigating the cause of a mysterious disease of tobacco, independently described an unusual agent that caused tobacco mosaic disease. This agent was later named *Tobacco mosaic virus* (TMV) [1]. During this period, viruses including *Potato virus X* (PVX), *Potato virus Y* (PVY) and *Lettuce mosaic virus* (LMV) were described. These viruses could be distinguished based on their transmission and method of disease induction. In addition, numerous techni‐

Viruses are among the most agriculturally important groups of plant pathogens, causing serious economic losses in many major crops by reducing yield and quality. A virus can be defined as a set of one or more nucleic acid template molecules, often encased in a protective coat of protein or lipoprotein, which is able to organise its own replication only within suitable host cells [1]. Because the genetic information encoded by viral genomes is limited, viruses depend entirely on host cells to replicate their genome and produce infectious progeny. Both plant and animal viruses can be classified according to the type of nucleic acid that makes up their genome. In plants, the vast majority of viruses have positive-sense (+) RNA genomes (i.e., the RNA genome has the same polarity as cellular mRNA), although negative-sense (−) RNA and double-stranded RNA viral genomes also exist. Other plant viruses have DNA genomes; the DNA can be double-stranded (caulimoviruses) or single-stranded (geminiviruses) [1, 2]. Cell-to-cell movement of plant viruses occurs through cytoplasmic "bridges" between cells

> © 2013 Costa et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Mechanisms for Survival**

Rodrigo Kazuo Makiyama,

http://dx.doi.org/10.5772/56253

**1. Introduction**

Alessandra Tenório Costa, Juliana Pereira Bravo,

Alessandra Vasconcellos Nunes and Ivan G. Maia

Additional information is available at the end of the chapter

ques for the study of viruses were developed.
