SEMINARS IN MOLECULAR MICROBIOLOGY

VIRAL LATENCY: STRATEGY OF IMMUNE EVASION

Viral Infections of Humans - An Introduction:

Acute infections: These are infections that are of relatively short duration with rapid recovery.

Examples include most viruses that infect humans, such as those that cause routine respiratory infections (e.g. cold viruses, influenza viruses) and gastrointestinal infections (e.g. Rotaviruses, Noroviruses). 

Persistent infections: These are infections in which the viruses are continually present in the body.

Some of the persistent infections could be slow infections that are characterized by having a long preclinical period (months or even years), or having late complications following an acute infection. An example is the subacute sclerosing panencephalitis (SSPE) that can follow an acute measles infection, and progressive encephalitis that can follow rubella. Another example of slow infections is those infections caused by prions (although prions are not considered typical viruses).

Other persistent infections are known as latent viral infections. These infections are characterized with periodic reactivation, and are a common outcome after acute infection with viruses, including the Herpes family viruses such as HSV-1, HSV-2, and VZV.

I. Viral Latency:

Viral latency is the ability of a pathogenic virus to lie dormant within a cell, usually denoted as the lysogenic part of the viral life cycle. A latent viral infection is known to be a type of persistent viral infection, and is distinguis-hed from a chronic viral infection.

In a viral latent infection, virus production ceases, and the virus remains in equilibrium with the host for long periods of time before symptoms again appear, but the actual viruses cannot be detected until reactivation of the disease occurs. In case of chronic virus infections, the virus can be demonstrated in the body at all times and the disease may be present or absent for an extended period of time. Examples of chronic infections include hepatitis B (caused by HBV) and hepatitis C (caused by HCV). 

The most common example of latent viruses is the Herpes virus family, Herpesviridae, all of which establish latent infections. Herpes viruses are a leading cause of human viral disease, second only to influenza and cold viruses. They cause overt disease such as cold sores and chickenpox, or they may remain latent for many years to be reactivated in later life, as in shingles.

Since the viral genome is not fully eradicated from the body of the host, a latent virus can be reactivated and begin producing large amounts of viral progeny without the host being infected by new outside virus, (denoted as the lytic part of the viral life cycle), and stays within the host indefinitely.

Latency is distinguished from lytic infection; in lytic infection many Herpes virus particles are produced and then burst or lyse the host cell. Lytic infection is sometimes known as "productive" infection. Latent cells harbor the virus for long time periods, and then occasionally convert to productive infection which may lead to a recurrence of Herpes symptoms.

Virus latency vs. Clinical latency: Virus latency should not be confused with clinical latency. Clinical latency is one of the things that occurs during the incubation period of an infection, in which the causative agent is present in the body and multiplying, but not causing symptoms. The virus involved in clinical latency is not dormant, as is the case with latent infections, but fully active and causing problems for the host organism.

Latency in Bacteriophages:

Latency in case of Bacteriophages is known as lysogeny. The lysogenic cycle is one of two phases of viral reproduction (the lytic cycle is the other). Lysogeny is characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome. The newly integrated genetic material, called a prophage can be transmitted to daughter cells at each subsequent cell division, and a later event (such as UV radiation) can release it, causing proliferation of new phages via the lytic cycle.

Examples of Latent Viral Infections:

An example of a latent virus is the HSV which may reactivate from latency from time to time in the ganglia and may spread to peripheral tissues to cause reactivated disease or asymptomatic virus shedding in the cornea, lips, genitalia, or other sites. Latent HSV-1 infection of the nervous system becomes increasingly prevalent among humans with age and most commonly involve the trigeminal ganglia, whereas involvement of other ganglia of the PNS is somewhat less frequent. HSV-2 primarily establishes latency in sacral ganglia.

Simian virus 40 (SV40) is a DNA virus that produces a lytic infection in the kidney cells of the African green monkey (these cells are used to cultivate viruses in the lab), but a latent infection in the cells of humans, mice, rats, and hamsters. Like lysogeny in bacteria, the SV40 genome becomes incorporated in the DNA of its host (in chromosome 7 in human cells). Although a human cell with harboring SV40 shows no outward sign of the virus, its presence can be detected by: (!) the appearance of viral-encoded antigens in the host cell, and (2) the ability of these cells to cause a lytic infection in African green monkey cells when fused with them.

In humans, lytic infections of plasma cells by the Epstein-Barr virus (EBV) occur in mononucleosis, whereas latent infections of B cells by EBV predispose the person to lymphoma. Also lytic infections by the human papillomas virus (HPV) cause genital warts, whereas latent infections by some strains of HPV lead to cervical cancer.

Viral latency as a mechanism of immune evasion:

The most important strategy HSV uses for immune evasion is the establishment of latent infection. In order to achieve a long-term persistence in the infected host, a virus must avoid killing too many host cells (which would lead to the early death of the host) and must avoid elimination by the host immune system. Latent infection of neurons allows HSV to escape elimination by the immune system and to persist indefinitely in the host.

Until quite recently, there appeared to be simple explanation why HSV latency is a successful strategy for viral persistence: it could be argued that suppression of viral replication and a lack of expression of viral genes - associated with the lytic cycle during latency - limits viral cytotoxicity and allows for the survival of the infected neurons for decades, and that because no viral protein expression occurs during latency, the immune system doesn't recognize and therefore cannot eliminate the latently infected cells.

However, there is now accumulating evidence that mechanisms by which HSV achieves long-term persistence in neurons involve a more complex pattern of HSV gene expression than previously thought. Latency is generally maintained by viral genes expressed primarily during the latent period, known as the latency-associated genes.

II. Mechanisms of Viral Latency:

A. Episomal latency:

Episomal latency refers to the use of genetic episomes during latency. An episome is a portion of genetic material that can exist independent of the main body of genetic material (the chromosome). In this type of latency, viral genes are floating in cytoplasm or nucleus as distinct objects, both as linear or lariat structures.

Examples of viruses with episomal latency:

1. The chicken-pox virus and herpes simplex viruses (HSV-1, HSV-2), which establish episomal latency in neurons, and leave linear genetic material floating in the cytoplasm.

2. The Gamma Herpesvirinae subfamily, which is associated with episomal latency established in cells of the immune system, such as B-cells in the case of Epstein-Barr Virus.

Advantages of episomal latency include the fact that the virus may not need to enter the nucleus, and hence may avoid ND10 domains from activating interferon via that pathway.

Disadvantages include more exposure to cellular defenses, leading to possible degradation of viral gene via cellular enzymes. Episomal latency is more vulnerable to ribozymes or host foreign gene degradation than provirus latency.

B. Proviral latency:

Proviral latency occurs when the virus genome integrates into the host genome, effectively becomes a provirus. This requires that the viral gene get into the nucleus and insert itself into the host genome.

The family of which exemplifies this behavior being the Retroviruses. For example, when a retrovirus HIV invades a host cell, the genomic RNA of the retrovirus is transcribed into DNA by the reverse transcriptase, then become inserted into the host genome by an integrase, and remains within the hosts own gene.

A provirus not only refers to a retrovirus, but is also used to describe other viruses that can integrate into the host chromosomes, another example being adeno-associated virus. It is thought that provirus may account for approximately 8% of the human genome in the form of inherited endogenous retroviruses.

Advantages include an automatic host cell division that results in replication of the viruses' gene. The provirus does not directly make new DNA

copies of itself while integrated into a host genome in this way. Instead, it is passively replicated along with the host genome and passed on to the original cell's offspring; all descendants of the infected cell will also bear proviruses in their genomes. Another advantage is the fact that it is near impossible to remove an integrated provirus from an infected cell without killing the cell.

Disadvantages include the need to enter the nucleus (and the need for packaging proteins that will allow for that) and increased difficulty in maintaining the latency.

Latent reservoirs of HIV; An obstacle to the eradication of virus:

In the field of HIV research, proviral latency in specific long-lived cell types is the basis for the concept of one or more viral reservoirs, referring to locations (cell types or tissues) characterized by persistence of latent virus. Specifically, the presence of replication-competent HIV in resting CD4-positive T cells, allows this virus to persist for years without evolving despite prolonged exposure to antiretroviral drugs. This latent reservoir of HIV may explain the inability of antiretroviral treatment to cure HIV infection.

Endogenous Retroviruses (ERVs):

A certain type of latency could be ascribed to the endogenous retroviruses. These viruses have infected human germline cells and have been incorporated into the human genome in the distant past, and are now passed through reproduction. Endogenous retroviruses can persist in the genome of their host for long periods. Generally these types of viruses have become highly evolved, and have lost the expression of many gene products. Some of the proteins expressed by these viruses have co-evolved with host cells to play important roles in normal processes.

Endogenous retroviruses, however, could be "infectious" after integration if they acquire 'knockout' mutations during host DNA replication. They can also be partially excised from the genome by a process known as recombinational deletion. Some human ERVs have been implicated in certain autoimmune diseases and cancers.

III. Maintaining Viral Latency:

How do latent viruses maintain their latency?

Both proviral and episomal latency may require maintenance for continued infection and fidelity of viral genes. Latency is generally maintained by viral genes expressed primarily during latency. Expression of these latency-associated genes may function to keep the viral genome from (1) being digested by cellular ribozymes, or (2) being found out by the immune

system. Certain viral gene products (RNA transcripts such as non-coding RNAs and proteins) may also inhibit apoptosis or induce cell growth and division to allow more copies of the infected cell to be produced.

A. Maintaining latency in HSV-1 infections; Latency Associated Transcripts (LAT):

The most common example of latency-associated gene products is the Latency Associated Transcripts (LAT) of the Herpes simplex virus. LAT is a length of RNA which accumulates in cells hosting long-term, or latent, Human Herpes Virus (HHV) infections. While these infected host cells would ordinarily undergo an organized death or be removed by the immune system, the consequences of LAT production interfere with these normal processes. LAT is expressed by the latent virus, and functions to: (!) regulate the viral genome, and (2) maintain viral latency by interfering with apoptosis through the down-regulation of a number of host factors, including Major Histocompatibility Complex (MHC) and inhibiting the apoptotic pathway.

Maintaining latency through inhibition of apoptosis:

In order to maintain a reservoir of latently infected host cells, Herpes virus interferes with apoptosis. During latency, most of the Herpes DNA is inactive, with the exception of LAT, which accumulates within infected cells. The region of HHV DNA which encodes LAT is known as LAT-DNA. After splicing, LAT is a 2.0-kilobase transcript (or intron) produced from the 8.3-kb LAT-DNA. The DNA region containing LAT-DNA is known as the Latency Associated Transcript Region.

Expression of LAT reduces the production of proteins involved in the apoptosis mechanism, including proteins caspase-8 and caspase-9. LAT expression also results in the suppression of herpes lytic genes. In rabbit trigeminal ganglia, extensive apoptosis occurred with LAT (-) virus but not with LAT (+) viruses. In addition, a plasmid expressing LAT blocked apoptosis in cultured cells. Thus, LAT promotes neuronal survival after HSV-1 infection by reducing apoptosis.

B. Maintaining latency in EBV infections; Epstein-Barr nuclear antigen 1 (EBNA-1):

For a virus to survive, it must elude the ever vigilant immune sentinels of its host. A latent virus can escape immune detection if it resides in non-dividing cells and doesn’t produce any proteins. No viral proteins means no red flags for immune cells. If the virus targets one of the many cell types that rarely divide, it’s relatively safe while latent.

But some viruses, like the gamma-herpesvirus, infect B cells of the immune system, which occasionally divide. The gamma-herpesvirus genome persists as circular pieces of DNA called episomes. When an infected B cell divides, the latent gamma-herpes virus episome must replicate and segregate

into daughter cells along with the cell’s genome. Viral replication and segregation requires the services of a protein called the episome maintenance protein.

The episome maintenance protein produced by EBV is known as the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1). This viral protein is essential for (1) replication of the episomal EBV DNAs, and (2) maintenance of latency.

Role of EBNA-1 in viral replication: EBNA-1 plays an essential role in replication and partitioning of viral genomic DNA during latent viral infection. During this phase, the circular double-stranded viral DNA undergoes replication once per cell cycle and is efficiently partitioned to the daughter cells. EBNA1 activates the initiation of viral DNA replication through binding to specific sites in the viral latent origin of replication, oriP. Additionally, it governs the segregation of viral episomes by mediating their attachment to host cell metaphase chromosomes.

Maintaining latency through evading immune response:

Although episome maintenance proteins might be a potentially recognizable target for immune cells, it was discovered that EBNA-1 harbors an amino acid element in its epitope (the region that binds to a T cell and triggers an immune response) that helps the viral protein evade the killer T cells that could destroy it. As a result of this, EBNA-1 is not recognized by the cellular immune system.

Lab studies show that the amino acid element limits EBNA-1’s interaction with T cells by inhibiting synthesis and, to a lesser degree, degradation of the protein. This results in impairing antigen processing and MHC class I-restricted antigen presentation, thereby inhibiting the CD8-restricted cytotoxic T cell response against virus-infected cells.

How this evasive action works or helps the virus in a living organism is not entirely clear. But if T cells aren’t presented with bits of viral protein, they have no way of knowing the virus is present.

IV. Outcomes of Viral Latency:

(A) Viral Reactivation:

While viral latency exhibits no active viral shedding nor causes any pathologies or symptoms, the virus is still able to reactivate via external activators (i.e. sunlight, stress) to cause an acute infection. In case of Herpes simplex virus, which generally infects an individual for life, a serotype of the virus reactivates occasionally to cause cold sores.

The sores are quickly resolved by the immune system, however may be a minor annoyance from time to time.

A | Infection of epithelial cells in the mucosal surface gives rise to productive replication, resulting in the production of progeny virions, which can spread to infect additional epithelial cells. Virus enters innervating sensory neurons, and nucleocapsids are transported to the neuronal cell body. The viral DNA is released into the neuronal nucleus and circularizes. Circular viral DNA persists in the neuronal cell nucleus, and the latency-associated transcript is expressed.

B | Upon reactivation, viral lytic gene expression is initiated, and newly formed capsids are transported to the axonal termini. Infectious virus is released from the axon and infects epithelial cells, resulting in recurrent infection and virus shedding.

Latent infections can also become a serious problem when a patient becomes immunocompromised, as the latent infection may manifest when the patient's immune system passes a critical point.

(B) Host Cell Transformation:

A more serious outcome of a latent infection could be the possibility of transforming the host cell, and forcing it into an uncontrolled cell division. This is usually seen in infections with human papilloma virus, in which persistent infection may lead to cervical cancer as a result of cellular transformation. Although it is unclear how high-risk HPV types cause cancer, studies indicate that malignant transformation involves the viral E6 and E7 gene products, which may exert their effect by inter-fering with the cellular proteins that regulate cell growth.

In vitro, cervical epithelial cells with integrated HPV-16 genes multiply faster than those with extrachromosomal (episomal) HPV-16. An explanation for this is that the expression of viral genes E6 and E7 is increased in cells with where the HPV-16 genome is integrated, and these gene products, oncoproteins E6 and E7, respectively bind and inactivate cell tumor suppressor proteins p53 and pRB.

Another example is the Epstein-Barr virus (EBV), which is a human gamma herpes virus that is best known for being the causative agent of infectious mononucleosis in man. A fascinating feature of this virus is its ability to persist in the host and it is estimated that more than 95% of adults are carriers of the virus. Importantly, EBV can transform latently infected primary cells from healthy individuals into cancerous ones, thereby causing important human cancers such as B-cell neoplasms (e.g. Burkitt's lymphoma and Post-transplant lymphomas), certain forms of T-cell lymphoma, and some epithelial tumors (e.g. gastric carcinomas).

Understanding viral latency, what triggers viral reactivation and the mechanism of transformation of normal host cells into malignant cells are critical for the development of strategies for the prevention and control of this intriguing virus and related cancers.

Epstein - Barr virus:

Epstein-Barr virus (EBV) is the causative agent of Burkitt's lymph-oma in Africa (as shown in figure), nasopharyngeal cancer in the orient and infectious mononucleosis in the west. It was first discovered as the causative agent of Burkitt's lymphoma

and it was later found that patients with infectious mononucleosis have antibodies that react with Burkitt's lymphoma cells.

Epidemiology and Mode of Transmission - A large proportion of the population (90-95%) is infected with Epstein-Barr virus, and these people, although usually asymptomatic, will shed the virus from time to time throughout life. The virus is spread by close contact, and can also be spread by blood transfusion.

Viral Targets - EBV only infects a small number of cell types that express the receptor for complement C3d component (CR2 or CD21). These are certain epithelial cells (oro- and naso-pharynx) and B lymphocytes. This explains the cellular tropism of the virus.

Life cycle - Virus infection involves two types of cells: (1) B cells, where infection is predominantly latent and has the potential to induce growth-transformation of infected cells; and (2) Epithelial cells, where infection is predominantly replicative. EBV latent infection of B-lymphocytes is necessary for virus persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva.

Following primary infection of B cells, a chronic virus carrier state is established in which the outgrowth of EBV-transformed B cells is controlled by an EBV-specific cytotoxic T-lymphocytes. Because of the marked CD4+ and CD8+ T-cell response to EBV nuclear proteins in Latency III infected B-

lymphocytes, EBV associated lymphoid malignancies are most common in immune compromised people.

Latently infected B cells can become permissive for lytic EBV infection. Infectious virus released from these cells can be shed directly into the saliva or might infect epithelial cells and other B cells. In this way a virus-carrier state is established that is characterized by persistent, latent infection in circulating B cells and occasional EBV replication in B cells and epithelial cells.

REFERENCE BOOKS AND WEBPAGES: Latency Strategies of Herpesviruses:

http://books.google.com.eg/books?id=wHrTa1E-r-AC&hl=en&source=gbs_navlinks_s http://en.wikipedia.org/wiki/Virus_latency http://student.ccbcmd.edu/courses/bio141/lecguide/unit3/viruses/virinf.html http://textbookofbacteriology.net/themicrobialworld/Herpes.html http://en.wikipedia.org/wiki/Lysogenic http://en.wikipedia.org/wiki/Provirus http://science.jrank.org/pages/2548/Episomes.html http://www.answers.com/topic/slow-virus-infection http://www.bio.miami.edu/~cmallery/150/gene/18x5.jpg http://lib.bioinfo.pl/pmid:10688801 http://www.horizonpress.com/ebv2 http://en.wikipedia.org/wiki/HHV_Latency_Associated_Transcript http://www.sciencedaily.com/releases/2005/03/050328173659.htm http://www.wikigenes.org/e/gene/e/3783709.html http://en.wikipedia.org/wiki/Epstein-Barr_nuclear_antigen_1 http://www.ncbi.nlm.nih.gov/pubmed/7957053 http://www.uniprot.org/uniprot/P03211 http://en.wikipedia.org/wiki/Endogenous_retrovirus http://www.nature.com/nrmicro/journal/v6/n3/fig_tab/nrmicro1794_F1.html http://en.wikipedia.org/wiki/File:Large_facial_Burkitt's_Lymphoma.JPG http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Viruses.html#LatentViruses http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TumorSuppressorGenes.html#hpv http://www.biologynews.net/archives/2005/04/10/

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