Although we may think of a virus as having characteristics of a living microbe, it is not alive. The virion particle is a very large biochemical complex consisting of a nucleic acid genome protected and carried within either a protein shell, termed a capsid, or within a membrane envelope. (Although prions were considered atypical viruses at one time, they are rogue, infectious proteins and not viruses.) The virion particle resembles a robotic spy. The genome, like a computer memory, can take different forms, as long as the genetic information can be stored, extracted, and used. Computer memories may be permanent and become part of the computer, like a hard drive, or be temporary, like a floppy disk. Similarly, viral genomes may be the more permanent DNA (either linear, circular, single, or double stranded) or can be single- or double-stranded RNA. DNA genomes can establish themselves in the nucleus as part of the cell, whereas RNA genomes are more temporary and most RNA viral genomes work from the cytoplasm of the cell.
The outer structures of the virus are a package that protects the genome, delivers it from host to host, and provides the means for interacting with and entering the appropriate target cell (Fig. 1). Capsids are protein shells usually in symmetric icosahedral or icosadeltahedral shapes (soccer balls). Like hardboiled eggs, these containers are impervious to drying, acids, and detergents. In contrast, enveloped viruses are surrounded by a membrane consisting of phospholipids and proteins, which must stay wet and can be disrupted by detergents and harsh environments including the acids and bile of the gut.
THE MISSION BEGINS
The innovative spy/saboteur takes advantage of the tools, machinery, and raw materials that are available within the host but must supply the plan and any tools that are not provided. Similarly, a virus must encode within its genes the structural components of the virus and any essential protein or enzyme that the cell cannot provide. For example, all RNA viruses must encode an enzyme that copies RNA from RNA (RNA-dependent RNA polymerase). The larger viruses, such as the herpes viruses, have the genetic capacity to set up a more sophisticated operation because they can encode a more extensive tool kit to facilitate their take over of the cell and establish their presence within the host. These tools may include enzymes such as a DNA polymerase to make new genomes, scavenger enzymes like thymidine kinase, to provide substrates for DNA synthesis, and proteins that facilitate growth in specialized cells or promote escape from host protections.
The start of the mission, whether spying or infecting, is begun by sneaking into the host. Most viruses, and I am sure, most spies, use a mode of entry that is readily available and that will provide the best cover. The least suspicious and most available means of entry for a spy would be public transportation. For most viruses, this would equate to eating and breathing. On special occasions, the spy may require a special insertion mechanism, for example, midnight delivery by submarine, whereas a virus might require direct injection into the bloodstream, as can be performed by a mosquito or other arthropod.
Once in the body, the virus must find and enter the appropriate target cell (Fig. 2). The choice of the appropriate target cell is important because not all cells have the machinery that can produce virus or allow the virus to establish a hiding place (latent infection) within the body. Finding and binding of the virus to the cell is mediated by the surface structures of the naked capsid virus or by the glycoproteins that decorate the envelope. These viral attachment proteins are the keys that interact with cell surface receptors and open the doors of entry into the cell. Some of these structures are like skeleton keys and bind to many different cell types, whereas others are very specific for certain cell types. A canyon on the surface of the rhino- or poliovirus (picornaviruses) acts as a keyhole that can be filled by specific receptors, which are members of the immunoglobulin family of glycoproteins. The glycoproteins on the Epstein-Barr virus bind to the receptor for the C3d component of complement (CD21), identifying the B lymphocyte as the prime target for the virus. The HIV gp120 glycoprotein picks out the CD4 molecule to target T-cells and myeloid cells [dendritic cells (DCs) and macrophages] and can switch target cells by mutating and changing the portion of the gp120 that binds to either a chemokine receptor present on myeloid cells and activated T-cells (CCR5) or 1 that is present on other T-cells (CXCR4).
Entry into the cell is truly spy-like. The virus either sneaks into the cell or tricks the cell into opening its doors and conveying the virus into the cytoplasm. Binding of the picornavirus receptor to the canyon on the virion surface opens the capsid structure and creates a hydrophobic portal for injection of the viral genome through the membrane into the cytoplasm. Some enveloped viruses, including herpes, retroviruses, and paramyxoviruses, will fuse their membrane envelope with the plasma membrane of the cell to deliver their genome into the cell. Many viruses bind to receptors on the cell surface, which trigger the internalization of the virus by receptor-mediated endocytosis, the same pathway that cells use to take up transferrin or low density lipoprotein. Although one would think that the normal acidification of the endosomal vesicle would be detrimental to a virus, viruses use this as a trigger to enter the cytoplasm. Acidification activates the fiber protein of the adenovirus capsid to lyse the vesicle and break into the cytoplasm. Acidification changes the confirmation of the glycoproteins of enveloped viruses, such as influenza or rabies virus, to promote fusion of their membrane with the vesicular membrane of the cell and deliver their genome into the cytoplasm.
VIRAL MACROMOLECULAR SYNTHESIS
Once inside the target, it seems that the first thing a spy does is to break into and alter the host computer, at least that is what Jennifer Garner and friends do on the television show, "Alias." Similarly, viruses must insert their genomic information into the cell's machinery and promote the synthesis of viral mRNA and protein. Each spy has their own way of doing this depending upon the task at hand.
The first step in mRNA synthesis for DNA viruses, except poxviruses, is to deliver their genome to the nucleus where it can be mistaken for cellular DNA. The sequences of the viral genes contain the same genetic passwords as cellular DNA so that they can use the cell's machinery for making and processing mRNA. RNA will be made by the host DNA-dependent RNA polymerase. Processing of the RNA goes through the same steps as for cellular mRNA: removal of introns (if present), addition of a 3′ polyadenosine tail, and attachment of the 5′ methylated (7-methylguanosine) cap, the structure that binds to the ribosome for protein synthesis. All of these modifications make the viral mRNA look and act like cellular mRNAs. Poxviruses are the DNA virus exception because they replicate in the cytoplasm. The poxviruses provide their own molecular tools for mRNA synthesis making them independent of the nuclear machinery.
Most DNA viruses divide protein synthesis into early and late phases. The proteins made during the early phase convert the cell into a more efficient factory for replicating virus. Once the cell has been subverted and new viral genomes are being produced, then the late genes are turned on and the cell churns out large amounts of viral structural proteins and glycoproteins to make the capsid or envelope proteins and glycoproteins. Activation of the late genes and synthesis of the structural proteins of a DNA virus is a death sentence for the cell.
Most RNA viruses do their business in the cytoplasm where there is easy access to the ribosome for protein synthesis. However, the cell has no machines (polymerases) to replicate RNA or make an mRNA from an RNA genome. By using RNA as the genetic information carrier, these viruses must encode and/or carry their own RNA-dependent RNA polymerases within the virion particle. In addition, these viruses must develop a way to acquire a 5′cap or an alternative way to bind to the ribosome for protein synthesis.
The viruses with a genome that looks like mRNA (positive sense) (except retroviruses) can immediately bind to ribosomes and initiate protein synthesis. The resulting protein cleaves itself into the individual viral proteins. Viruses with a negative sense RNA (like a photographic negative) cannot be deciphered by the cell's ribosome. The genome of these viruses is decorated by enzymes (RNA-dependent RNA polymerase), which will convert the genome into individual mRNAs for each of the viral proteins. Influenza viruses are an exception because their proteins are encoded by individual segments of negative-stranded RNA and these segments are converted into mRNA in the nucleus. Influenza steals the 5′cap from cellular mRNAs and the 3′ polyadenosine is attached before leaving the nucleus so that their viral mRNAs will look like cellular messenger RNAs. The REO and rotaviruses have a segmented double-stranded RNA genome surrounded by enzymes (inner capsid), which will convert the negative strands of the genome into mRNA with 5′ cap.
Although the retroviruses, including HIV, have a positive sense RNA genome, they carry a reverse transcription enzyme that converts the RNA genome into DNA and then another viral protein promotes the integration of the viral DNA into the host chromosome. The integrated retroviral DNA becomes a permanently "embedded" spy. Viral mRNAs and even new copies of the virus can then be transcribed just like any other cellular gene.
Once the virus has manipulated the cell into making mRNA, the viral proteins are synthesized by the cell's ribosome and glycoproteins are processed by the same machinery as cellular glycoproteins. Especially during the late phase of protein synthesis, the cellular machinery is likely to be devoted to production of viral proteins to the exclusion of cellular proteins.
The large amounts of protein that are produced to build the virion particles places a stress upon the cell that sets off a molecular alarm. This alarm leads to the placement of a block in protein synthesis (see below). Some cells, such as neurons, are more sensitive to this stress than other cells because they do not have the synthetic or metabolic machinery to support the process without going into starvation. The cell's response to the unreasonable demand for so much protein synthesis is to shutdown the factory. On a molecular basis, this is done by phosphorylation of a key element in the initiation of protein synthesis, the elongation initiation factor-2-alpha (eIF2a). Excess unfolded or misfolded protein in the endoplasmic reticulum activates a protein kinase (PERK), which phosphorylates the eIF2a. Phosphorylation of eIF2a prevents the assembly of the large ribosomal subunit with the mRNA and small ribosomal subunit and this prevents protein synthesis. Without protein synthesis, the virus cannot replicate and the mission is blocked.2
The production of large amounts of a limited number of viral proteins also results in the production of large amounts of proteinaceous trash, which is processed into peptides, which are displayed on the cell surface in MHC I (HLA) molecules as a signal to killer CD8 T-cells. Specific viral and cellular proteins or improperly folded proteins become marked by the attachment of ubiquitin for degradation by the proteosome. This proteolytic machine degrades the proteins into small peptides, which are delivered into the endoplasmic reticulum through the transporter associated with processing (TAP). Some of these peptides will bind to the heavy chain of the MHC I molecule, trigger the attachment of its beta-2-microglobulin component, and accompany the MHC I molecule to the cell surface. The peptide-filled MHC I molecule is the signal for cytotoxic CD8 T-cells to bind and convince the target cell to commit suicide (apoptosis). This eliminates the viral factory and prevents it from being used to advance the viral mission. The immunologic processes are described in more detail in the previous article in the Basic Microbiology and Immunology section in Infectious Diseases in Clinical Practice entitled, "Vaccines Make Good Immune Theater: Immunization as Described in a Three Act Play"3 and below.
Completion of the viral mission requires that each virus replicate their genomes. For DNA viruses, the new genome is replicated like cellular DNA, in a semiconservative manner. The smaller DNA viruses stimulate and convince the cell to grow and make DNA (including viral DNA) and also remove their inhibitions to growing (inactivated by p53 or RB growth suppressor proteins). The larger viruses encode their own tools for viral replication, a viral DNA polymerase and other enzymes, which provide more independence from the cell and help speed up the replication of the genome. For the retroviruses, the cell's DNA-dependent RNA polymerase is "duped" into making the new genome as if it were making a mRNA. The new genome is just a full-length RNA copy of the integrated viral genome. Hepatitis B virus does something similar to the retroviruses. The cell makes a longer than full genomic length RNA, which gets reverse transcribed by the viral enzyme into a DNA genome. As a result, hepatitis B virus can be inhibited by lamivudine, a reverse transcription inhibitor that is also effective against HIV.
Our cells have set a trap for retroviruses and hepatitis B virus, viruses that use a reverse transcription. Many cells, especially lymphocytes and macrophages, express a family of mutator proteins, the apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family, which convert deoxycytidines to deoxyuracils. For our cells, these enzymes make beneficial somatic mutations in cellular genes. For example, they change a single nucleotide within the apolipoprotein B protein gene to create a stop codon that generates a new shorter form of ApoB with a different function. However, as HIV is being assembled, the APOBEC3 binds to the viral RNA genome and its nucleocapsid (gag) protein and converts most of the deoxycytidines to deoxyuracils as the negative DNA strand is copied from the viral RNA by the reverse transcription. The resulting cDNA is converted into genetic gibberish and the virus cannot replicate. A similar consequence awaits the hepatitis B virus.4
Most RNA viruses replicate their genome using an RNA-dependent RNA polymerase that is carried into the cell or encoded by the virus and made soon after infection. This polymerase makes an RNA template from the genome and uses it to synthesize positive or negative sense RNA genomes. During some point in the production of an mRNA or a new genome, a double-stranded RNA structure will be generated between the template and the new copy.
The double-stranded RNA replicative intermediate that is formed during replication of the RNA virus genome is so unusual to the cell that it sets off several viral burglar alarms. The double-stranded RNA replication intermediates provide pathogen-associated molecular patterns, which bind to toll-like receptors within the cell, and especially DCs, to activate innate cytokine and cellular responses. The double-stranded RNA replication intermediate is also the strongest trigger for induction of interferon (IFN) production and activation of Type 1 IFN-related protections.
Type 1 IFNs (alpha or beta) are produced in a virus-infected cell and then released to act as an early warning system to surrounding cells. IFN is a very potent antiviral agent and the binding of one IFN molecule to its cell surface receptor sets off a molecular cascade that puts the cell on alert in the "antiviral state." A molecular booby trap is set up for the virus that consists of inactive forms of another protein kinase for eIF2alpha, called protein kinase R (PKR), and a polymerase (oligoadenylate synthetases; OAS) that makes an unusual polyadenylate activator of RNAse L. The trigger for both the PKR and the OAS is double-stranded RNA, which strings multiple copies of these enzymes together to activate them. Ultimately, the combination of degradation of mRNA by the RNAse L and the inhibition of the ribosome by PKR phosphorylation of eIF2a results in inhibition of protein synthesis and a block in viral replication. If the inhibition of protein synthesis lasts long enough, then the cell will commit hara-kiri (apoptosis).5
IFNs (alpha and beta) can also activate innate immune responses including natural killer cells.
Ultimately, all of the parts of the virus must be assembled into the virion particle so that it can move to new target cells and hosts. At first thought, the process seems to be analogous to assembling a jigsaw puzzle by shaking the parts in a box. However, the process is not as random as the shaken puzzle box. For some capsid viruses, individual proteins associate into larger and larger complexes that eventually become empty capsid shells. The large number of viral proteins within the cell promotes enough collisions between the proteins to drive the process to completion. Many of these structures are built on scaffolds made of cellular membranes. Once the capsid shell is assembled, it is filled with the genome. Alternatively, the capsid proteins assemble around or associate with the genome. For enveloped viruses, the protein-coated genomes (nucleocapsid) associate with viral proteins inserted into or associated with the cellular membrane and the nucleocapsid wraps itself in the membrane.
Virus release separates the viral spies from the viral saboteurs. Most capsid viruses act as viral saboteurs. They accumulate in the cytoplasm and must convince the cell to blow apart to be released. Lysis of the cell may be a consequence of toxic viral proteins, the build up of viral material, an inability to rebuild cellular structures or induction of apoptosis or necrosis. Most enveloped viruses are subtler and acquire their envelope from the plasma membrane as they push their way out of the cell without killing the cell. Herpes viruses are released by several mechanisms, including vesicular transport mechanisms similar to those used to release cellular proteins and cell lysis.
Upon release, the viruses can spread to other cells and expand the infection. Some viruses (eg, herpes simplex virus) can sneak through the back alleys between cells (intercellular spaces) to infect neighboring cells without exposing themselves to antibody detection. Other viruses (HSV, varicella-zoster, measles and other paramyxoviruses, and HIV) recruit large numbers of cells into their factory by fusing them together into multinucleated giant cells (syncytia).
Ultimately, the virus must seek out new hosts to continue its mission. The route of transmission is determined by the tissue that replicates the virus. Respiratory secretion requires replication in the lung whereas fecal-oral transmission requires replication in the mucosal epithelium of the pharynx or the intestines. Transmission in blood, by a mosquito or in a transfusion, requires that the virus infect blood cells or establish a sufficient viremia and be in the bloodstream when the needle or the arthropod takes its blood.
The means of virus spread are also very dependent upon the structure of the virus. As a rule, most naked capsid viruses are very hardy and can endure drying, acid environments, and even detergents. Naked capsid viruses can be passed by fecal-oral and most other means and will resist soap and detergent treatment (except rhinoviruses). For example, the hepatitis A virus can even withstand the gastrointestinal tract and sewage systems. In contrast, enveloped viruses, viruses that have a membrane around them, must stay wet and are disrupted by acids and detergents that will disrupt the envelope (except coronaviruses and hepatitis B). These viruses are transmitted in aerosol droplets, in blood, transplanted organs, and secretions.
Sexual transmission requires an additional criteria. Sexually transmitted viruses have to be especially sneaky spies because in addition to replication on the genitalia or within secretory cells, the virus must be transmissible before or in the absence of symptoms. "Having a headache," aches, pains, lesions, or other symptoms are a deterrent to transmission of sexually transmitted diseases.
As soon as the virus enters the host, it is in a race to finish its mission to establish the infection and spread to other cells and hosts. The virus must accomplish its mission before it is detected by the host and counterespionage measures are activated. As indicated above (in italics), the alarms are set off by viral protein synthesis and the replication of the viral genome and these alarms can ring throughout the infected individual. To prevent establishment of the initial base of operations, the individual cell activates a block in protein synthesis that will prevent production of virus. The block is established by phosphorylation of eIF2a and degradation of mRNA. In addition, these cells send out an IFN warning signal to surrounding cells to set up the same type of block upon sensing virus replication. As an additional hindrance to the viral mission, the cell surface expression of viral antigenic peptides in MHC I molecules mark the infected cell for destruction by cytotoxic T-cells. As a last ditch attempt to stop the progression of the virus, the infected cell may commit apoptosis, either due to the block in protein synthesis or induced by cytotoxic CD8 T-cells. Oftentimes, this is sufficient to stop the progression of the virus and the viral incursion will receive minor or no symptomatic notice.
The successful virus, like a good spy, has evolved molecular tools and tricks to escape detection and undermine or evade the surveillance and defensive measures of the host. Some of these molecular tools are genetically stolen from the host and imitate a normal cellular function and some others are developed by trial and error (mutation) and may be unique to the virus.
The more saboteur-like viruses replicate very quickly and accomplish their mission before the host can set up its barriers. Polio, for example, replicates very quickly in the oropharynx, the cells lyse and virus enters the blood stream or gets dumped into the gut to be spread throughout the body or to other people by the fecal-oral route. In this case, the innate, inflammatory, and immune responses activated by the host in an attempt to stop the virus and clean up the mess are usually too late to stop the spread of the virus.
Viruses that establish persistent infections are especially good spies. Hepatitis C virus, cytomegalovirus, and other viruses have an extensive molecular toolkit and are adept at overcoming the roadblocks, traps, and hazards that the host sets up to stop and eliminate viruses. The specific molecular tools that HCV, CMV, and other viruses use to overcome the cellular blocks to replication and evade the innate and immune protections of the host are presented below (see Tables 1 and 2).
Manipulation of the Infected Cell
Phosphorylation of eIF2a
Viruses have evolved several different tricks to evade the consequences of phosphorylation of eIF2a. Phosphorylation of eIF2a prevents protein synthesis from mRNAs with 5′caps. The easiest evasion approach, taken by picornaviruses (poliovirus, coxsackie virus, echovirus, and rhinovirus) and other positive sense RNA viruses, is to initiate protein synthesis without eIF2a. These viruses take a back door entry into the ribosome that is used by some cellular mRNAs to make protein. A loop of the viral RNA directly interacts with the ribosome through an internal ribosome entry site, the ribosome assembles and viral protein synthesis proceeds. Poliovirus also sabotages the rest of cellular protein synthesis by destroying the ribosomal protein that recognizes and binds to the 5′cap on the cellular mRNAs.
Some viruses avoid the protein synthesis roadblock by preventing it from being applied.2,5 Several viruses have proteins that prevent the phosphorylation of eIF2a by neutralizing the PKR and PERK enzymes. Influenza A is especially tricky and activates a cellular inhibitor of PKR. Hepatitis C (NS5A protein), vaccinia virus (E3L, K3L), and even HIV (tat) make proteins that are competitive inhibitors of PKR, which prevent the enzyme from tagging the eIF2a with phosphate. Adenovirus (VAI RNA) and Epstein-Barr virus (EBER RNA) make small double-stranded RNAs, which bind to the PKR to prevent it from being activated by other double-stranded RNAs. Herpes simplex virus has two ways to deal with the phosphorylation of eIF2a: a protein (US11) that binds and inhibits PKR and another protein (ICP34.5) that promotes the removal of the phosphate from eIF2a.2 A region of the ICP34.5 protein resembles a cellular protein that binds to and activates the cellular protein phosphatase 1 to remove the phosphate from eIF2a.6 In so doing, the ICP34.5 short circuits the protection that is attempted by PERK or PKR.
As a last ditch effort to prevent the replication of the virus, the cell will become apoptotic. For most viruses, this must be prevented7-9 because dead cells cannot replicate viruses. By preventing PKR from blocking protein synthesis, the virus also blocks molecular signals that initiate apoptosis. Once initiated, viruses can stop the cascade with an analogue of a cellular inhibitor of the induction of apoptosis (BCL-2) or by inhibiting the caspase enzyme cascade that immediately precedes the death sentence. Epstein-Barr virus (BHFR-1 protein), human herpes virus 8 (HHV8; Kaposi sarcoma virus) (KSBcl-2 protein), and adenovirus (E1b 19K protein) produce analogues of BCL-2. Once initiated, caspase inhibitors are produced by adenovirus (E3/14.7) and poxviruses (CrmA) to stop the onset of cell suicide. Herpes simplex virus produces a protein kinase (US3) that inhibits apoptosis by an unknown mechanism.
The IFN system was specifically designed to foil the virus in its attempts to complete its mission. Viruses evade the consequences of the IFN system by preventing the IFN from being made, by taking out of commission the trigger proteins that initiate the elaborate cascade of events that establish the antiviral state in the cell, or by ignoring or stepping around the blocks established by the enzymes of the activated antiviral state that is induced by IFN (such as the PKR described above).10-13
By binding to double-stranded RNA, the NS1 protein of influenza A silences the best inducer of IFN production. HHV8, the virus that causes Kaposi sarcoma, and human papilloma virus encode proteins that interrupt the pathway that induces IFN synthesis. After IFN is produced, the poxviruses are ready with viral encoded decoy receptors that bind the IFN and prevent the IFN from initiating the antiviral response in the infected cell. Hepatitis C virus, measles, and other viruses compromise the JAK-STAT pathway of protein phosphorylation events that follow IFN binding to its receptor to prevent the expression of the proteins of the antiviral state.
APOBEC3 is a formidable defense against retroviruses.4 It sneaks aboard new viruses and converts new cDNAs into genetic gibberish. HIV devotes one of its proteins to defend against this threat. The vif protein neutralizes the threat from the APOBEC3 protein by preventing it from being incorporated into virions. Vif binds to the APOBEC3 protein and tags it for degradation by the proteosome. If there is no APOBEC3, there is no threat to the fidelity of the cDNA.
MHC Expression of Viral Peptides to Cytotoxic T-cells
The production of large amounts of viral proteins in the infected cell is an unavoidable risk of detection that viruses must take to make new virus. This is a risk that most spies would avoid because it calls attention to the viral-infected cell, in this case, by the CD8 T-cell.
The cell surface of every nucleated cell is decorated with peptides representative of the proteins made in the cell (host and viral), displayed in an MHC I (HLA) molecule as identification markers to killer CD8 T-cells. CD8 T-cells either ignore (tolerance) or lack (T-cells recognizing self are eliminated in utero) the ability to recognize peptides representative of self but activated CD8 T-cells will trigger apoptosis in cells expressing foreign peptides.3
The best way a virus can evade CD8 T-cells is to avoid detection. When Epstein-Barr virus goes into hiding (latency) after an episode of mononucleosis, it only needs to express 1 protein, the EBNA 1 (Epstein-Barr virus nuclear antigen 1), and this protein completely lacks the potential to generate peptides that can bind or be presented by human MHC I molecules.13 Other viruses have developed ways of preventing MHC I molecules from acquiring viral peptides or the display of the peptide-loaded MHC I molecules on the cell surface.14,15 The herpes simplex virus (ICP47) and cytomegalovirus (US6) encode proteins that prevent uptake of peptides through the TAP channel into the endoplasmic reticulum, and as a result there are no peptides to bind to MHC I molecules. Interestingly, the HSV ICP47 protein evolved for humans and the ICP47 protein does not work in mouse cells. Adenovirus E19 protein and cytomegalovirus US3 protein prevent the assembly of the MHC I-peptide complex by binding to components of the molecular scaffold (tapasin) that is used in the process. Human CMV also promotes the degradation of MHC I proteins before it is filled with peptides through the action of its US2 and US11 proteins.
Once the identity and the presence of the intruder is known, whether the intruder is a spy or a virus, then specific defenses can be developed. These defenses are directed at both the agent and its hiding place. Our bodies target the virion particle with antibody-mediated responses, like smart bombs, and target cell-mediated responses against the infected cell. Viruses have developed numerous ways to evade these immune responses. These processes of evasion can be grouped into 3 mechanisms: (a) remaining invisible (replication in an immunoprivileged site or establishing a latent infection), (b) changing appearance (antigenic variation), or (c) inactivation of the defense mechanism (inactivation of DCs or T-cells). Each of these approaches will be briefly described.
Remaining or Becoming Invisible to the Immune Response
The good spy uses many tricks to remain hidden from detection. Similarly, viruses have several ways to remain hidden from the immune response. They can keep a low profile, spread to new cells in ways that are inaccessible to the immune response, or they can infect immunoprivileged sites, like the eye or the CNS, where cell-mediated immunity is limited.
The herpes viruses wait out the immune response in a hiding place (latency) until the heat is off (depression of the immune response) and then resume production of virus (this type of spy would be called a mole). For example, cytomegalovirus and varicella-zoster virus remain in latency until immune surveillance levels drop and then they reestablish their mission, replicate, and potentially move to new hosts.
Herpes simplex virus and other viruses can spread to other cells without being exposed to antibody by being released into cell-cell junctions or by fusing cells together into 1 large multinucleated giant cell. Rabies virus jumps into hiding within neurons (an immunoprivileged site) soon after infection where it cannot be acted upon by host protections. Rabies then travels to and replicates in the brain and salivary glands. The virus even promotes its transmission because the rabid animal is more likely to bite and hence spread the virus in its saliva.
Changing Appearance (Antigenic Variation)
Once the host has realized that it has been infected, an immunologic all person's bulletin goes out to detect and eliminate the virus. Protective antibodies are generated, which are very specific for a surface characteristic of the virus, an exposed region of a viral capsid protein or an envelope glycoprotein. To evade antibody recognition, some viruses use the same approach as spies; they use many different disguises or change their appearance.
HIV facilitates its spread in the presence of antibody by putting on camouflage stolen from DCs. After binding of HIV to DCs, the virus is endocytosed into vesicles that are then released by the cell as exosomes. These HIV containing vesicles efficiently deliver the virus into T-cells and cannot be blocked by antibody.
Unlike spies, however, most viruses change their appearance by trial and error (mutation) at the expense of the less successful viruses (selection). Most viruses are prone to mutation and the presence of antibody will eliminate the viruses that remain recognizable. The viruses that escape detection will be selected and will continue the viral mission.
Rhinoviruses exist in so many different serotypes (disguises) that it is difficult to develop antibodies that will protect against future infections. Influenza, hepatitis C, and HIV are notorious for changing their antigenic appearance due to the error prone nature of their viral polymerases. For example, the HIV polymerase generates approximately 5 mutations per genome. Changes in the HIV gp120 protein occur during the persistent infection of the individual to sidestep the antibody threat and can even change the coreceptor specificity of the virus (CCR5 to CXCR4) to change the target cell that gets infected. The antigenic mutational drift of influenza A strains is 1 reason for updating annual flu vaccines.
Cell-mediated immune responses are much more difficult to fool than antibody responses. Unlike antibody, which can select for a viral variant, CD8 T-cells are directed at changes in the infected cell and, specifically, the peptide trash that is displayed on the cell surface by MHC I molecules. The peptide trash that is displayed is representative of the population of viruses within the cell and not individual viruses. Influenza and HIV can send in a new team of virus variants unknown to the previously established cell-mediated immune responses. The large changes associated with a shift in influenza strains or infection with a different clade of HIV would require the development of a new T-cell response.
Inactivation of the Immune Defense Mechanism
Viruses use several spy-like approaches to inactivate antibody-mediated defenses. Spies keep the enemy agents busy following decoys and they confuse the enemy's counterspy devices. Similarly, hepatitis B virus causes the infected liver cell to make decoy particles consisting of the hepatitis B surface antigen (HBsAg). These decoys overpower the antibody response to the virus. Herpes simplex virus has a glycoprotein (gE) that binds to the Fc portion of immunoglobulin G that inactivates the antibody, and the bound antibody may also provide camouflage for the virus and infected cell. The Fc portion is the part of the antibody that would interact with complement or phagocytic cells to facilitate the clearance of the virus. Binding of the HSV glycoprotein C to the C3 component of complement would similarly inactivate this important immune effector molecule. Binding of IgG and C3 by gE and gC can also camouflage the virus with a coat of host proteins.
Viruses can evade cell-mediated immune responses by using subterfuge, tricking the immune cells, and by "knocking off" important immune cells. Epstein-Barr virus encodes an analogue of interleukin 10, a cytokine that antagonizes (prevents activation) the protective type of cell-mediated immune response. Measles virus infects and inhibits the proper maturation of the DC. Maturation of the DC is essential for the presentation of antigen to CD4 and CD8 T-cells, the activation of these cells, and the proper initiation of an immune response.11,16 HIV can also infect the DC but it uses the ultimate approach to escape immune control by depleting the numbers of CD4 T-cells. CD4 T-cells produce the cytokines that activate and control most aspects of the immune response, other CD4 T-cells, CD8 T-cells, B-cells, macrophages, and DCs.
The viruses that still plague humans have the tools to succeed in their mission. They use their viral molecular toolbag to sneak in and subvert the protections that humans have developed to amplify their numbers and spread through the population to new receptive hosts. Some viruses have a bigger molecular toolkit than other viruses and some viruses are sneakier than other viruses.
Mounting the defense against the invasion of these molecular spies is not without consequences. Microbial disease that results from infection is a combination of the pathology induced by the microbe and (as described in the "Microbial Diseases, Are They Self Inflicted?"1) host-induced immunopathology. A key determinant in the amount of pathology caused by either mechanism is the extent to which the infection progresses before being controlled. The further a virus spreads within a host, the more rigorous the host response that will be necessary to resolve the infection and the greater the potential for immunopathogenesis. Just as with counterespionage, the best defense against a virus is the ability to recognize the interloper and then limit the replication and spread of the agent within the body and within the population, immune preparedness. For this reason, vaccination of the individual and the population is so important. More on vaccines and the nature of the immune response can be read in the previous edition of IDCP in "Vaccines Make Good Immune Theater: Immunization as Described in a Three Act Play."3
Unfortunately for the human race, the smallpox virus has used a spy/saboteur's ultimate weapon and played on the human emotions of distrust, greed, and thirst for power to continue its existence. Smallpox would have been eliminated by an effective program of quarantine and vaccination except that stockpiles of the virus were amassed as a bioweapon by the former USSR. Unfortunately, other viruses are also able to use this same weapon to promote their development as bioweapons. We can only hope that appropriate political, immunologic, and antiviral roadblocks will be effective against these infectious spies and saboteurs.
I would like to thank Angelo De Lucia, Colleen Sowick, Neena Goel, and others for reading through the drafts of this manuscript.
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2. Mohr I. Neutralizing innate host defenses to control viral translation in HSV-1 infected cells. Int Rev Immunol
3. Rosenthal KS. Vaccines make good immune theater: immunization as described in a three-act play. Infect Dis Clin Pract
4. Cullen BR. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol
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