Despite their simplicity relative to bacteria, viruses still possess various biochemical targets for potential attack by chemotherapeutic agents. An appropriate chemical compound may interrupt each of these. Hence, a thorough understanding of the specific biochemical events that occur during viral infection of the host cell should guide the discovery of site-specific antiviral agents. The process of viral infection can be sequenced in seven stages:
1. Adsorption, attachment7 of the virus to specific receptors on the surface of the host cells, a specific recognition process.
2. Entry, penetration7 of the virus into the cell.
3. Uncoating, release7 of viral nucleic acid from the protein coat.
4. Transcription, production of viral mRNA from the viral genome.8
5. Translation, synthesis8 of viral proteins (coat proteins and enzymes for replication) and viral nucleic acid (i.e., the parental genome or complimentary strand). This process uses the host cell processes to express viral genes, resulting in a few or many viral proteins involved in the replication process. The viral proteins modify the host cell and allow the viral genome to replicate by using host and viral enzymes. The mechanisms by which this occurs are complex. This is often the stage at which the cell is irreversibly modified and eventually killed.
6. Assembly of the viral particle. New viral coat proteins assemble into capsids (the protein envelope that surrounds nucleic acid and associated molecules in the core) and viral genomes.8
7. Release of the mature virus from the cell by budding from the cell membrane or rupture of the cell and repeat of the process, from cell to cell or individual to individual.8 Enveloped viruses typically use budding on the plasma membrane, endoplasmic reticulum, or Golgi membranes. Nonenveloped viruses typically escape by rupture of the host cell.
Life cycles of different viruses provide many different possible schemes. To show the typical complexity, the life cycle of the influenza type A virus is shown in Figure 9.1.
Step 1: The virion attaches to the host cell membrane via hemagglutinin and enters the cytoplasm via receptor-mediated endocytosis, forming an endosome. A cellular trypsinlike enzyme cleaves HA into products HA1 and HA2 (not shown). HA2 promotes fusion of the virus envelope and the endosomal membranes. A minor virus envelope protein M2 acts as an ion channel, thereby making the interior of the virion more acidic. As a result, major envelope Ml dissociates from the nucleocapsid.
Step 2: In Step 2, viral nucleoproteins are translated into the nucleus by interaction between the nucleoproteins and cellular transport machinery. Step 3: In the nucleus, the viral polymerase complexes transcribe (Step 3a) and replicate (Step 3b) the viral RNAs. Step 4: Newly synthesized mRNAs migrate to cytoplasm where they are translated.
Step 5: Posttranslational processing of HA, NA, and M2 includes transport via the Golgi apparatus to the cell membrane (Step 5b). Structural regulatory proteins and nuclear export proteins move to the nucleus (Step 5a). Step 6: The newly formed nucleocapsids migrate into the cytoplasm in a nuclear export protein-dependent process and eventually interact via M1 with a region of the cell membrane where neuraminidase, hemagglutinin, and M2 have been inserted. Step 7: The newly synthesized virions bud from the infected cell. Neuraminidase destroys the sialic acid moiety of cellular receptors, thereby releasing the progeny virions.
The initial attachment of viral particles to cells probably involves multiphasic interactions between viral attachment protein(s) and host cell surface receptors. For instance, in the case of the alphaherpesviruses, internalization involves a cascade of events that involve different glycoproteins and different cell surface molecules at different stages. Different cell surface proteins may be used for the initial attachment and entry into target cells and for cell-to-cell spread across closely apposed populations of cells.9 The pattern of systemic illness produced during an acute viral infection in large part depends on the specific organs infected and, in many cases, on the capacity of the viruses to infect discrete populations of cells within these organs. This property is called tissue tro-pism.10,11 The tissue tropism of a virus is influenced by the interaction between various hosts and viral factors.
Although the specific viral attachment proteins and specific receptors on target cells are important, a variety of other virus-host interactions can play an important role in determining the tropism of a virus. Increasing attention is being focused on coreceptors in mediating viral binding. For instance, entry of HIV-1 into target cells requires the presence of both CD4+ and a second coreceptor protein that belongs to the G-protein-coupled seven-transmembrane receptor family, including the chemokine receptor proteins CCR5 and CXCR4. Cells that express CD4+ but not the coreceptor are resistant to HIV infection. Host cellular receptors can be integrins, heparans, sialic acids, gangliosides, ceramides, phospholipids, and major histocompatibility antigens (to name a few). There is substantial evidence that the cellular receptor for influenza viruses is the peptidogly-can component N-acetylmuramic acid, which binds a protein molecule, hemagglutinin, projecting from the viral surface.12 The binding of N-acetylmuramic acid and hemag-glutinin sets in motion a sequence of events, whereby the viral envelope and the host cell membrane dissolve into each other, and the viral contents enter the cell. Initiation of HIV-1 infection involves the interaction of specific glyco-protein molecules (gp120) that stud the viral cell surface with an antigenic CD4+ receptor molecule on helper T lymphocytes along with a cytokine coreceptor.13-16 Substantial evidence indicates that viruses enter cells by endocytosis, a process that involves fusion of the viral envelope with the cell membrane, intermixing of components, and dissolution of the membranes of virus and cell. Various receptors and coreceptors facilitate this reaction.17-19
Before a virus can begin a replication cycle within a host cell, its outer envelope and capsid must be removed to release its nucleic acid genome. For complex DNA viruses such as vaccinia (its binding receptor is the epidermal growth factor receptor), the uncoating process occurs in two stages17:
1. Host cell enzymes partially degrade the envelope and capsid to reveal a portion of the viral DNA, which serves as a template for mRNA synthesis.
2. mRNAs code for the synthesis of viral enzymes, which complete the degradation of the protein coat, allowing the virus to fully enter the host.
The proteins of the viral envelope and capsule are the primary targets for antibodies synthesized in response to immunization techniques. Protein synthesis inhibitors such as cycloheximide and puromycin inhibit the uncoating process, but they are not selective enough to be as useful as antiviral agents.
In the critical fourth and fifth stages of infection, the virus usurps the energy-producing and synthetic functions of the host cell to replicate its own genome and to synthesize viral enzymes and structural proteins.20 Simple RNA viruses conduct both replication and protein synthesis in the cytoplasm of the host cell. These contain specific RNA polymerases (RNA replicases) responsible for replication of the genome. Some single-stranded RNA viruses, such as poliovirus, have a (+)-RNA genome that serves the dual function of messenger for protein synthesis and template for the synthesis of a complementary strand of (—)-RNA, from which the (+)-RNA is replicated. In poliovirus (a picornavirus), the message is translated as a single large open reading frame whose product is cleaved enzymatically into specific viral enzymes and structural proteins.18,21 Other RNA viruses, such as influenza viruses, contain (—)-RNA, which serves as the template for the synthesis of a complementary strand of (+)-RNA. The (+)-RNA strand directs viral protein synthesis and provides the template for the replication of the (—)-RNA genome. Certain antibiotics, such as the rifamycins, inhibit viral RNA polymerases in vitro, but none has yet proved clinically useful. Bioactivated forms of the nucleoside analog ribavirin variously inhibit ribonucleotide synthesis, RNA synthesis, or RNA capping in RNA viruses. Ribavirin has been approved for aerosol treatment of severe lower respiratory infections caused by RSV.
Retroviruses constitute a special class of RNA viruses that possess an RNA-dependent DNA polymerase (reverse transcriptase) required for viral replication. In these viruses, a single strand of complementary DNA (cDNA) is synthesized on the RNA genome (reverse transcription), duplicated, and circularized to a double-stranded proviral DNA. The proviral DNA is then integrated into the host cell chromosomal DNA to form the template (apovirus or virogene) required for the synthesis of mRNAs and replication of the viral RNA genome. During the process of cDNA biosynthesis, a ribonu-clease (RNase) degrades the RNA strand, leaving only DNA. Oncogenic (cancer-causing) viruses, such as the human T-cell leukemia viruses (HTLV) and the related HIV, are retro-viruses. Retroviral RT is a good target for chemotherapy, being inhibited by the triphosphates of certain dideoxynucle-osides, such as 2',3'-deoxy-3'azidothymidine (AZT, zidovudine), 2',3'-dideoxycytidine (ddCyd, zalcitabine), and 2',3'-dideoxy-2',3'-didehydrothymidine (D4T, stavudine), all of which have been approved for the treatment of AIDS. The nomenclature of these agents is straightforward. A 2',3'-dideoxynucleoside is referred to as ddX, whereas the unsaturated 2',3'-dideoxy-2',3'-didehydronucleosides are named d4X. The dideoxynucleoside triphosphates are incorporated into viral DNA in place of the corresponding 2'-deoxynucle-oside (i.e., 2'-deoxythymidine, 2'-deoxycytidine, or 2'-de-oxyadenosine) triphosphate.22,23 This reaction terminates the viral DNA chain, because the incorporated dideoxynucleo-side lacks the 3'-hydroxy l group required to form a 3',5'-phosphodiester bond with the next 2'-deoxynucleotide triphosphate to be incorporated.
The DNA viruses constitute a heterogeneous group whose genome is composed of DNA. They replicate in the nucleus of a host cell. Some of the DNA viruses are simple structures, consisting of a single DNA strand and a few enzymes surrounded by a capsule (e.g., parvovirus) or a lipoprotein envelope (e.g., hepatitis B virus). Others, such as the herpesviruses and poxviruses, are large, complex structures with double-stranded DNA genomes and several enzymes encased in a capsule and surrounded by an envelope consisting of several membranes. DNA viruses contain DNA-dependent RNA polymerases (transcriptases), DNA polymerases, and various other enzymes (depending on the complexity of the virus) that may provide targets for antiviral drugs. The most successful chemotherapeutic agents discovered thus far are directed against replication of her-pesviruses. The nucleoside analogs idoxuridine, trifluridine, and vidarabine block replication in herpesviruses by three general mechanisms: first, as the monophosphates, they interfere with the biosynthesis of precursor nucleotides required for DNA synthesis; second, as triphosphates, they competitively inhibit DNA polymerase; and third, the triphosphates are incorporated into the growing DNA itself, resulting in DNA that is brittle and does not function normally. Acyclonucleosides (e.g., acycloguanosine) are bioactivated sequentially by viral and host cell kinases to the acy-clonucleotide monophosphate and the acyclonucleoside triphosphate, respectively. The latter inhibits viral DNA polymerase and terminates the viral DNA strand, since no 3'-hydroxyl group is available for the subsequent formation of a 3',5'-phosphodiester bond with the next nucleoside triphosphate. The structure of acyclovir with the acyclosugar chain rotated into a pentose configuration shows clearly the absence of the 3'-hydroxyl group.
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