The Human Spumavirus and Its Use as a Vector in Gene Therapy
This paper will focus on the human spumavirus (HSV), or foamy virus, and its usefulness in gene therapy, including some background on gene therapy and the virus itself. In turn, it is essential that a background of retroviruses be given since spumaviruses are a class of retroviruses and retroviruses are also used in gene therapy. This paper will begin with an introduction to gene therapy, retroviruses, spumaviruses, and finally, discussion of an experiment that proves HSV is of particular interest as a vector in gene therapy.
Gene Therapy Overview
A gene in a human cell is a stretch of DNA that usually acts as a blueprint for making a particular protein by spelling out the amino acids needed to compose that protein. Thus, the gene is a length of DNA that encodes for a particular protein. The gene is made up of multiple copies of connected nucleotides adenine, thymine, cytosine, and guanosine (the four building block/nucleotides that make up DNA) and encodes for complementary messenger RNA, which is composed of the same four nucleotides only instead of thymine there is its compliment in RNA: uracil. All cells in the body carry the same genes in the chromosomes of their nuclei.2 Each cell produces only the selected genes that it needs and copies them into messenger RNA, which then is used as a template to construct the cells required proteins. If a gene is mutated, its protein product may not be made at all or may work too poorly or too aggressively.2 This flaw can disturb vital functions of cells and tissues that use the normal protein and therefore cause symptoms of disease.2
Gene therapy itself is the use of nucleic acids (DNA particles) as therapeutically useful molecules.2 The most obvious application of this is to correct defects in genetically inherited diseases (monogenic disorders) such as cystic fibrosis.2 A normal gene can be delivered to a cell that has a mutated gene it physically takes the place of the flawed version of the gene on the chromosome.2 Most uses of gene therapy add a useful gene into a selected cell type to compensate for a missing or ineffective version of a particular gene or to instill an entirely new property; this is gene replacement.2 Abnormal cell behavior is often the result of an altered gene that has either absent or unregulated expression. A mutation in one gene can cause the cell to malfunction by directing the synthesis of a dysfunctional protein that can lead to abnormal, or no, cell function.2 Such diseases as sickle cell anemia, adenosine deaminase deficiency, cystic fibrosis, and muscular dystrophy result from a mutated protein and can be corrected by gene replacement.2 Some cancers can also be fixed this way since some genetic mutations cause a cell to divide uncontrollably, as cancers do.4 Another application is gene transfer, which is literally taking a gene from one organism and inserting it into another organism's genetic material.2 This can be used to stimulate an immune response, to mediate specific cell killing, or to produce a molecular decoy required for viral replication. Gene therapy can also alter the gene directly allowing the patient to produce a sustained amount of the normal protein and avoid daily pills or painful injections.2 The two other methods of gene therapy used are gene addition and gene control.4 Gene addition leads to the insertion into the cell of a new gene that enables the production of a protein not normally expressed by that cell.4 Gene control is another method and can alter or control the expression of a gene.4 The main hurdle for gene therapy however is that the gene of interest must be transferred into the nucleus with the rest of the DNA, not just into the cell.2
The vector is the vehicle used to introduce a gene into a target cell.2 Disabled viruses are frequently used because they can perform many tasks necessary to transfer a gene.2 The main task is the binding of the virus to a target cell and delivering the viral genome with the gene of interest into the nucleus for transcription. By integration of the virus into the host cells genome, the gene of interest becomes part of the host cell's DNA and since the virus is disabled, no disease occurs. Non-viral vectors, such as plasmid DNA (circular independent DNA molecules) found in bacteria are also used because they have no foreign proteins and can therefore avoid immunological problems common with viral vectors.2 There are several ways to go about delivering a therapeutic gene, but this paper will concentrate on the viral vectors. Vector selection and method of delivery can not be generalized because each disease can have specific requirements such as target tissue and amount of gene product required which dictates the vector and method to be used.2 Also, a vector's properties may or may not be desirable for a particular application.
Therapeutic genes are provided to patients in two ways. In both cases, the genes are first put into vectors that are able to deposit the foreign genes into cells.2 In the most common method (ex vivo), scientists remove cells from a selected tissue in a patient, expose them to the gene vectors in a lab, and then return the genetically corrected cells to the patient.2 Another method, in vivo, is where scientists introduce the vectors directly into the tissue being treated.2
The ideal gene delivery system is that which is able to enter a large amount of cells and integrate its DNA into the host cell's chromosomes.2 This is where retroviral vectors could be useful because they operate in this way. Members of the retrovirus family can be engineered to carry foreign genes into cells and splice them into that cell's chromosomes.2 To be used as vectors, the virus must be gutted of its genes, disposing of the genes that may be harmful to the host cell, while keeping those genes that enable the virus to integrate its DNA into the host chromosome.2 By attaching this retained part to the therapeutic gene, a retrovirus is created that is capable of infecting cells and splicing the corrective gene into the host cell chromosome permanently.2 This integrated gene will then be copied and passed on to all future cell generations. Also, one or more therapeutic genes (the new functional genes) may be substituted for viral genes responsible for viral replication and virulence.2
There are, however, some disadvantages to using retroviruses as vectors. The ability to integrate genes into chromosomes can pose a problem because there is no control over how many copies of the gene become integrated or where they are inserted.2 Integration is somewhat random with retroviral vectors so it is possible that the therapeutic gene could be inserted into another very important gene and disrupt its expression thus creating a new problem.2 A gene could also integrate into a regulatory region of a gene that is responsible for cell proliferation thereby possibly causing uncontrollable, cancerous growth and tumors.2 Another problem is that copies of the virus with the therapeutic gene could be taken up by cells that are not intended to receive the gene and therefore decrease the amount of transfer to the target cells and even cause adverse physiological effects.2 Current retroviral vectors can reach chromosomes (which are in the nucleus) only when the nuclear membrane surrounding the nucleus of the host cell dissolves; this occurs only during cell division.2 On the other hand , other vectors have the advantage here because they do not have this restriction. What must be done is that a patient's dysfunctional cells be removed and replication/division stimulated so that the retroviral vector can be inserted.2 Once this is done, the cells can then be returned to the patient.2
Some of these problems are currently being researched and corrected.2 To increase specificity of the retrovirus vector and direct it to a particular cell type, the viral envelope proteins can be altered to be specific for this particular cell type.2 As will be explained, these proteins are specific to receptors on the surface of other cells. Once recognized by the host cell, the virus binds to the host cell's receptors and fuses with its outer membrane and injects the new genetic material. Finally, HIV (a retrovirus) vectors are being investigated because they have genes that code for proteins that transport genes to the nucleus.2 If these genes can be inserted into a retrovirus that does not cause human disease, retroviruses may then have a way of working as vectors in cells without the restriction of required host cell division.2
Overview of Retroviruses
The human spumavirus (HSV)/human spumaretrovirus (HSRV), or foamy virus, is a unique retrovirus that was first discovered in the 1950s and is still an enigma.5 The virus's apparent lack of pathogenicity leads one to believe that it is not a huge concern to humans as a disease carrier. Yet, HSV is thought to be involved in chronic long-term illnesses such as chronic fatigue syndrome as well as neurodegenerative disorders and possibly even aging itself.5 Although a vector does not need to be nonpathogenic for the pathogenic genes may be removed, it would be much easier if there was no pathogenicity in the virus. A conclusion as to whether or not HSV causes disease of any kind has yet to be made. This paper will concentrate on some of the unique properties of the retrovirus, and more specifically the human spumavirus, which makes its use as a vector in gene therapy of particular interest.
The human spumavirus is a member of the genus Spumaviridae and the family Retroviridae. Retroviruses have always been strange, mysterious, and unique viruses but were never really recognized until the emergence in 1983 of HIV, the most famous of the retroviruses. They are so named because of their ability to reverse transcribe, or go from RNA to DNA instead of DNA to RNA as do most other viruses and all animal cell types. A general background of retroviruses help understand their usefulness as a viral vector in gene therapy as a whole.
A General Retrovirus Model
All retroviruses have various structures unique to themselves although they follow a similar general retroviral structure with little variation. All viruses are basically made up of protein and genetic material, DNA or RNA. Starting from the outside and moving inward (as shown in the above diagram), the outermost layer is a lipid envelope that is hydrophobic in nature (insoluble in water) and contains a large amount of transmembrane glycoproteins (TM) that emanate out of the envelope.6 Transmembrane glycoproteins are proteins with accessory sugar components that span the whole membrane or envelope of the virus. These are the proteins responsible for membrane fusion and each one is linked to a surface glycoprotein.6 The surface glycoprotein (SU) is responsible for binding the virus to its glycoprotein specific respective receptor on the cell membrane of the host cell.6 Following this binding, the fusion of the host cell and virus membranes occurs allowing the virus to dump its contents (genetic material and proteins) into the host cell. On the inside of this lipid envelope is the amorphous matrix (MA) made up of matrix protein.6 This matrix surrounds the capsid (CA) of the virus and merely serves as a spacer between the inside of the envelope and the capsid. The capsid is composed of the most abundant protein in the virus (capsid protein) making up approximately 33% of the total weight of the virus protecting the nucleocapsid core (NC) of the virus.6 The nucleocapsid is found inside the capsid and is made up of nucleocapsid protein. Inside the nucleocapsid core is found a single stranded RNA genome along with the proteins protease (PR), reverse transcriptase (RT), and integrase (IN).6 Protease is the protein essential for cleavage of the structural proteins.6 The proteins encoded by the gag gene (one of the genes found in all retroviruses) such as the matrix and capsid proteins are cut down by protease during the maturation stages, leading to the condensation of the core (closer packing of contents). Reverse transcriptase is the enzyme responsible for the actual reverse transcription of the RNA genome into DNA.6 Integrase is encoded by the pol gene (another gene found in all retroviruses) and is needed for the integration of the eventual viral DNA product into the DNA of the host cell, producing what is known as the provirus.6
The genome is the genetic material of an organism, be it DNA or RNA. All retroviruses have genomes consisting of two molecules of single stranded RNA (positive sense) like that of mRNA.6 Positive sense means it has the same organization as messenger RNA, the product of normal transcription (DNA to RNA); the RNA genome has the same polarity as the eventual RNA transcript. All retroviruses have three invariantly ordered genes: gag, pol, and env, in that order from the 5' end to the 3' end (shorthand for 5 prime and 3 prime).6 The ends are labeled this way because one end ends with a phosphate group to the fifth numbered carbon on the first ribonucleotide and the other end ends with a hydroxyl (OH) group attached to the third numbered carbon of the last ribonucleotide. The viral RNA genome actually encodes for these genes and once these genes are transcribed, the result is an mRNA product from this transcription. The process of translation will then produce the mRNA's respective proteins, those proteins for which the genes of mRNA encode. The gag gene encodes for the capsid core proteins, the pol gene encodes for reverse transcriptase and protease, and the env gene encodes for the envelope antigens (surface proteins).6 Some retroviruses have additional genes and as will be shown, spumaviruses have three bel genes unique to themselves.
General Retrovirus Genome
There are a number of general features of the retroviral genome, starting at the 5' end of the genome and moving to the 3' end, in accordance to the above general model. Like a normal RNA, the 5' end begins with a 7-methylguanosine cap (a modified protective ribonucleotide: a guanosine with attached methyl group) that simply protects it from digestion by enzymes.6 The R-U5 and U3-R components mark the ends of the genome (5' and 3').6 These are the ends to which the viral DNA will be integrated (attached to) the host cell DNA.6 Next is the U5 sequence, which is the first part of the genome to be reverse transcribed and will form the 3' end of the provirus genome which itself actually has the promoter elements previously mentioned.6 This section is essential for transcription of the DNA into RNA and then eventual translation of the RNA into protein. Following the U5 sequence is a rather important sequence called the primer binding site (PBS), which will bind a complimentary primer that starts reverse transcription.6 Without a primer, no kind of transcription can happen, reverse transcriptase can not start from scratch. The leader region is only needed once the RNA product from provirus transcription is translated into protein.6 It marks the beginning of the viral genes.6 It serves no other function. Following this are the three invariant genes gag, pol, and env.6 At the 3' end of the RNA genome, after these genes, is first a polypurine tract (PPT) made up of mostly purine nucleotides (adenosines and guanosines).6 It is responsible for initiating the DNA product complimentary to the viral RNA genome during reverse transcription.6 After this is the U3 region, which forms the 5' end of the provirus after reverse transcription.6 Again, this U3 region contains all of the promoter elements needed for transcription of the provirus DNA.6
The life cycle of the retrovirus is lytic in nature; meaning it undergoes a normal attachment, replication, assembly, etc. To initiate the infection (as shown below), the surface glycoprotein of the virus must bind to a specific surface receptor on the host target cell (attachment).6 Once this happens the virus can enter the host cell (entry). This penetration and uncoating of the virus is not fully understood but once inside there is at least a partial uncoating eventually leaving only the nucleocapsid and its contents in the cytoplasm.6 Reverse transcription is a complex process that takes place inside the cytoplasm of the host cell with the needed reactants of reverse transcriptase, RNA genome, and nucleotides free within the host cell cytoplasm.6
The Retroviral Life Cycle
In the next step of the retroviral life cycle seen above, the provirus DNA produced in reverse transcription migrates from the cytoplasm ("open space" of a cell) of the host cell to its nucleus where integration into the host cell DNA will take place.6 The enzyme integrase catalyzes the integration of the provirus into the DNA of the host cell.6 Integrase cleaves off both the ends of the provirus and host cell DNA target site.6 Then the actual integrase activity joins the ends of the provirus to the "new ends" of the host DNA.6 Eventually, the genome is replicated as cellular DNA with the rest of the host cell DNA eventually producing, through normal cellular transcription and translation, the host cell and viral proteins (expression & post-transcriptional regulation).
Finally, there is packaging of the viral genome. Virus particles will be assembled (assembly) in the cytoplasm from the translated proteins made from the integrated viral genes.6 The new viruses are released by budding out of the host cell.6 The genome will be packaged as the budding process takes place.6 Following budding, maturation of the new virus particles takes place through cleavage events catalyzed by protease and leading to the condensation of the core.6 These new virions then repeat the whole process over again.
Important Parts of a Retroviral Vector
Retroviral vectors are usually based on the Moloney murine leukemia virus model which is capable of infecting both mouse and human cells, thus enabling proper testing in a mouse model before being tested in a human model.7 The two viral genes gag and pol can be replaced by the transgene of interest (the functional gene to be introduced into the dysfunctional gene), mainly because they are non-essential and lack the packaging (psi) sequence essential for passing on of the viral vector to future host cell generations.7 The essential regions of the retrovirus include the U5 and U3 regions (which contain those essential promoter regions) and the psi sequence.7 The transgene expression can be driven by the promoter elements found in the U5 and U3 regions or by alternative viral or cellular promoters.7 Any alteration of the these regions can alter transgene expression because they have the promoter elements, which in effect turn the gene "on or off" meaning that it will cause the therapeutic gene to make its protein product or not. If these regions are destroyed, the promoter and the cell's ability to make the new functional gene/protein is also destroyed, possibly preventing the therapeutic gene from even working. As mentioned earlier, the envelope proteins of the retrovirus may be altered to enable the retrovirus to target a specific cell type.7 This is done by altering the env gene or its product thereby altering the envelope glycoprotein and what it specifically binds to on a target.7 This in turn can either widen or narrow the range of host cells that are targeted by the virus. In addition, one can replace the retrovirus env gene with that of another virus, which also widens the host cell range because it will target a completely different cell type.7
Human Spumaviruses as Distinct Retroviruses
A background on retroviruses explains the human spumavirus and what makes it so unique. Initial examination reveals the structural and virological differences of the human spumavirus. Overall, spumaviruses are spherical and have a ring shaped nucleoprotein core surrounded by a lipid bilayer membrane envelope.5 One distinct difference of HSV from other retroviruses discovered by electron microscope was that this envelope is covered by needle-like spikes and has a nucleocapsid that rarely condenses.5 These differences are unique to the spumavirus and distinguishes the spumaviruses from other retroviruses. As previously stated, the human spumavirus is also known as human foamy virus.5 The reason for the foamy name is that they cause a characteristic cytopathic effect in a tissue culture that leads to the lysis of host cells.5 This effect physically looks like foam bubbling outward. This causes in turn, a group of cells to take on the apperance of one huge multinucleated and multivacuolated cell; this is known as a syncytium.5 This also gives them another name, syncytium-forming virus.5 It is this foamy effect along with the foamy appearance under light microscopy that gives the spumavirus its name.5
The genome of the human spumavirus, compared to other retroviruses, is the same except for its unique bel genes. Like a normal retrovirus, HSV encodes for gag, pol, and env genes as well as the unique bel genes to be discussed.1 The bel genes are found after the env gene and extend into the 3' long terminal repeat.1 In addition, the life cycle of HSV is identical to that of the general retrovirus model. For future reference, it is important to note that the genes are always written in lower case and their consequent protein product is always capitalized. Questions to be answered in evaluating the use of HSV as a gene therapy vector are for what are the bel genes encoding and what proteins are produced from them.
The Human Spumavirus Genome
Above is a diagram of the HSV. Like a normal retrovirus, HSV has the gag, pol, and env genes described previously. The gag gene of HSV encodes for the three structural proteins: the matrix protein (MA), capsid antigen (CA), and nucleocapsid antigen (NC).1 The pol gene encodes for the enzyme proteinase (Pro), as well as reverse transcriptase (Pol), RNase-H (Rnh), and integrase (Int).1 The overall sequence is distinct from other retroviruses and actually closer to a virus known as "Moloney murine leukemia virus", yet the structural motifs for these HSV enzyme's functional activities is highly conserved as compared to other retroviruses.1 The env gene encodes for the transmembrane protein (TM) as well as the surface glycoprotein (SU).1
Following these genes are four genes unique to and only found in spumaviruses. First is the bel 1 gene, which will be discussed in detail later.1 Next is the unique chimeric bet gene.1 The chimeric name indicates that it is a combination of two genes or sections of genes. In this case, the bet gene is made up of amino terminal bel 1 sequences as well as the entire bel 2 sequence.1 Bet has no related proteins and is only found in spumaviruses.1 It is abundantly expressed in HSV infected cells.1 Bel 2 protein synthesis starts downstream of bet.1 Both Bel 2 and Bet proteins are localized in the cytoplasm of the host cell and have unknown function.1 As for the bel 3 gene, its sequence is found within the bel 2 gene but more distal from the 3' end.1
Finally, the questions remain as to exactly what the unique bel and bet genes of the human spumavirus do, or at least, what do bel 1 and bel 3 do. This is important because a viral vector is useful only if it causes no disease of any kind. For this to be possible, the proteins and the genes that make up the virus must be known, as well as whether or not they cause the disease. This would enable a scientist to cut out any harmful genes before use as a viral vector. Of course, HSV does not seem to cause any illnesses (it has no pathology), but long term effects or degenerative effects of the virus remain to be seen. Unfortunately, as will be discussed, bel 1 and/or bel 3 may be involved in any disease complex HSV may cause, if it causes any.
Experiments to Determine Function of Bel Proteins as Well as Pathogenicity of HSV
If HSV causes any disease complex (has pathogenicity) then the only cause of this would be the Bel proteins because it is the only part of HSV that differs from other retroviruses. The experiments described below were done to address this. It was found that bel 1 is a specific trans-activator of the HSV LTR as well as being required in replication of the virus.1 This means that Bel 1 protein will cause production of viral proteins through transcription/translation by binding to the U3 promoter regions. An experiment was done to see if this Bel protein could do this same thing to other retroviral U3 promoter regions and cause them to make their proteins.1 If it did, then the Bel 1 protein can indirectly lead to the other virus's disease occurring by causing the other virus to make its own proteins. In other words, a gene that would otherwise be dormant by itself may be activated by Bel 1 of HSV. The Bel 1 protein was injected into several separate mixtures of different retroviruses present in cells such as fibroblasts and human T-cells.1 In each case, the other virus was not producing any of its protein.1 It was found that Bel 1 could trans-activate the HIV-1 LTR promoter regions of the HIV-1 genome, causing it to make its proteins whether causing disease or not.1 This indicates that in patients with both viruses present, Bel 1 may activate HIV-1 transcription and may also contribute to its pathogenesis.1 In other words, an otherwise dormant HIV-1 virus is activated by HSV Bel 1 protein. When an individual is dually infected, HSV may be a cofactor in the other virus's pathogenesis and consequently have a hand in causing disease indirectly.1 Even though indirect, there is a chance that HSV could cause a disease of some kind, so for use in a viral vector, the bel 1 region must be cut out. Of course this leads to problems since the Bel 1 protein is also needed in replication and once an HSV vector is introduced it could only be used once. It is possible that a gene which makes a protein required for replication could be fused into the HSV genome once bel 1 is cut out so that the vector would replicate and be used over and over again, thus taking the place of the absent bel 1 region.
Probably the strangest aspect of HSV that yet remains a mystery is the bel 3 gene and its consequent protein, even though bel 2 and bet have unknown functions and are still being researched. In vitro, the Bel 3 protein was found to strongly stimulate human T-cells (a part of the human immune response), leading researchers to believe the protein acts as a superantigen and therefore giving HSV great pathogenic potential.1 Bel 3, like Bel 1, may also cause disease, especially if it stimulates a component of the human immune response. The other Bel proteins did not have this effect, suggesting that Bel 3's function may well indeed be that of a spumaviral superantigen and consequently be the cause of any disease or pathology that the human spumavirus may or may not have.1 Finally, the Bel 3 protein sequence is exactly the same as that of an HIV-1 protein called Nef, which is required itself for high virus numbers and full expression of symptoms and disease of HIV-1; research is currently being done here to determine if Bel 3 serves this same function.1 The bel 3 gene and Bel 3 protein still remain a mystery, as does HSV itself. As with bel 1, if these cause disease, bel 3 must be cut out of any HSV viral vector. There is no point to using something to correct disease if it causes another.
A number of experiments have been done on HSV recently to find out exactly what it does and whether this virus causes any kind of disease/infection be it long term or short term? It should be common knowledge that a virus generally causes some type of disease so one that does not seem to perform this function is odd and requires further insight. A recent study was done by Dr. John Martin who postulated that HSV was the direct viral cause of chronic fatigue syndrome (CFS).8 Chronic fatigue syndrome is characterized by severe fatigue, along with muscle and joint pain, and even loss of balance, coordination, arithmetic ability, and ability to recall words and phrases.8 Symptoms vary widely and are similar to other illnesses making CFS hard to diagnose.8 Some scientists even believe these symptoms to be a result of psychological problems, not CFS.8
Martin found HSV in the cerebral spinal fluid of ten patients who had severe neurological conditions that mimicked those of brain ailments like multiple sclerosis.8 He also found the human spumavirus in 160 of 300 patients (53.3%) diagnosed with CFS.8 He questioned whether this was a coincidence or whether HSV was the cause. The original ten patients had been referred by psychiatrists, neurologists, and rheumatoidologists and were suspected of having various neurological disorders, yet their symptoms did not quite fit any of these other neurological disorders such as MS, lupus, and encephalitis; they were closer to CFS.8 Dr. Martin was also able to grow and replicate HSV in a test tube using these patients' cerebral spinal fluid.8 Of course, even with all these studies there were still some problems. The referral patients with mild, moderate, and severe symptoms gave no consistency between the degree of sickness and presence of the virus.8 Also, the tests may not have been sensitive enough.8 Such tests would have to be very sensitive, because even a 53.3% HSV positive rate does not mean HSV is involved, one would need at least an 85% HSV positive rate to consider looking at HSV as a cause.8 In the end through further experimentation, it was found that the virus associated with CFS was in actuality a new form of Hantavirus never before seen.8
The neurodegenerative effects of HSV on mice may be of particular interest to this examination. Again, if the human spumavirus causes a disease, the particular genes/proteins that cause it must be eliminated for use as a viral vector. Another attempt by scientists to tie HSV to some kind of disease involved an experiment where modified versions of HSV were injected into unfertilized mice eggs.1 The first version contained a mutation that resulted in a non-functional integrase protein so that viral DNA could not be inserted into the host DNA.1 A second version had most of the gag and env genes deleted and completely lacked the pol gene.1 Another version only contained the bel genes.1 The common property between them all was that they all had intact bel genes so that they could only make Bel proteins. Again, experimenters were searching for the culprit of any disease caused by HSV. Of those mice that were born, all of the mice developed severe neurological symptoms at about two months of age.1 Those showing severe neurological diseases died within 4-6 weeks.1 Their tissues were examined once dead and all were found to have alterations in only the central nervous system and striated muscle of the spinal musculature.1 Bel 1 protein was detected in the affected tissues in large amounts.1 From this, the bel 1 gene was postulated to be the gene most likely responsible for the neuromyodegenerative diseases and symptoms found in the mice.1 This meant that if there is any disease caused by HSV, Bel 1 might be the primary cause. These observations in the mouse model show a potential for HSV to cause disease, especially with respect to neurodegenerative diseases. Still, the pathogenicity of HSV for its natural host (humans) also remains to be seen though extensive research is currently being done. Together with the CFS study, these two particular experiments directly show how HSV is most likely involved in some kind of disease, most likely those of a neurodegenerative manner. To use the virus as a viral vector in gene therapy it is essential that the gene/protein causing this be eliminated. From these studies, it seems that Bel 1 or 3 protein may be the culprit.
There are several reports tentatively linking the presence of HSV and certain neurological conditions, but the majority of naturally infected hosts do not display disease. HSV has been recovered from patients with de Quervain subacute thyroiditis as well as CFS, MS, lupus, and encephalitis, yet none of these isolates have been characterized well.1 It has been established that several different kinds of retroviruses induce neurologic disease or nerve cell lesions.1 Visna virus's demyelination in sheep (breakdown of the protective myelin sheath in nerve cells), HIV-1 AIDS dementia, and human T-lymphotropic virus (HTLV) associated myelopathy all stem from neurologic effects of retroviruses.1 These observations suggest that retroviruses causing neurodegenerative disease are not exceptions but the rule, and HSV may or may not be included in that rule. Overall, the reason for the apparent lack of pathology in those affected by HSV is not known, but it may suggest the virus is associated with long term chronic degenerative disease, most likely neurodegenerative ones. The neural tissue specific effects of HSV in mice are of great interest. Also of great interest is the role and mechanism of action of Bet, Bel 2, and Bel 3 proteins; some things are known about them but still nothing conclusive.1 Altogether, spumaviruses in general have a broad host range and also, because of the lack of pathogenesis in hosts, these viruses may be eventually used as retroviral vectors in gene therapy. Again though, the greatest interest in HSV lies in its role in the development and pathology of human disease, especially those of a neurodegenerative manner and how the bel and bet genes may be involved in this, if they are involved at all. This must be discovered before HSV can conclusively be used as a viral vector in gene therapy.
Properties of HSV Important in Its Use as a Viral Vector
This background explains how the mysterious human spumavirus (HSV) may be used as a retroviral vector in gene therapy. Human spumaviruses are emerging from obscurity and are being increasingly considered as potential vectors in gene therapy. The following study demonstrates that not only does HSV have a broad tropism (can infect many cell types) but the receptor for it is expressed on mammalian, avian, and reptilian cells giving it a broad host range as well.3 HSV is apathogenic in infected humans and animals meaning it does not seem to cause any disease. Of all the aforementioned genes of HSV, only bel-1 with its trans activating property is needed for replication and consequently, any therapeutic gene expression in a host cell.3 It may also be of interest to know that since HSV usually remains in the cell when replicating; it can not spread to other cells.3 The following short study, along with some previously mentioned properties, proves that there are some basic aspects of HSV that are relevant to its use as a vector in gene therapy. The part of the whole study that is of particular interest is how it was demonstrated that HSV could infect a wide variety of cell types as well as a wide variety of species.
In the study, twenty mammalian cell types were used which included six different kinds of human cell types from lung fibroblasts to epithelium along with cell types from mice, primates, rodents of various types, and pigs, two kinds of avian cells, as well as iguana epithelium.3 Inoculation of these various cell lines resulted in HSV infection detected by staining of the virus antigen with antibodies.3 It was found that more than half of these cell lines were readily infected by HSV and therefore had the appropriate HSV antigen receptor.3 The other half was also infected but not to the same degree as the rest.3 Those readily infected included four of the six human types (not epithelium), all of the mouse and avian types, hamster, and dolphin cell types.3 Those not infected as readily included two human epithelium cell types, pig cell types, and a monkey cell type.3 The remaining cells indicated an intermediate level of infection.3 This analysis of a wide variety of cell lines from diverse species confirms that the receptor for HSV is widely expressed in mammals and is also present in birds and reptiles. In addition, HSV has also been found to infect cell lines from rabbits, cows, dogs, cats, chickens, and sheep as well as many different human cell types including epithelial, neuronal, myeloid, and fibroblastoid.3 This ubiquitous nature of the HSV receptor suggests that it may perform an essential cellular function to eukaryotic (plant/animal) cells.3 Also, the ubiquitous presence of this receptor in diverse cell lines from both mammals and invertebrate indicates that it may not even be protein.3 This wide variety of targets for the HSV antigen proves that HSV based vectors could be quite useful for gene transfer in a wide variety of cell types.3 That fact in turn, makes this vector a likely candidate to be used for transfer of a needed gene to almost any cell type thereby fixing a wide variety of cellular problems therapeutically.3
In conclusion, there are several properties of the human spumavirus that are of interest to its development as a vector in gene therapy. One of these is obviously the virus's apparent lack of pathogenicity. This would make it easier to use because no time or money would need to be spent on developing a way to get rid of any harmful genes in HSV as had to be done with HIV. In addition, the patient would not have to worry about coming down with a disease later on in life after the problem the therapy corrected was finally gone. Still, as previously mentioned, there are considerable ongoing efforts to find out if HSV is indeed involved in long-term diseases or those of a neurodegenerative nature. Finally, as the above experiment proves, HSV is capable of infecting a wide variety of cell types and species therefore it can transfer a therapeutic gene to practically any cell with an abnormal gene and consequent abnormal cell function. The only value of its use in other species is for their use in a testing model situation. What is of greater interest is the fact that the virus can infect a wide variety of cell types in humans. Not only that, but the virus has been isolated from a wide variety of tissues in humans, in fact from practically every tissue in humans.5 This again increases its ability to correct any abnormal gene and thereby help cure a wide variety of inherited diseases such as MS, cystic fibrosis, sickle cell anemia, and many others either present or dormant in the body. Further studies are still being done on how useful this virus is in gene therapy and its possible pathogenicity. This is just the first step in determining whether or not this mysterious virus can be used in such a manner. But, it represents the beginning of possibilities for the human spumavirus, either as a vector in gene therapy or as a pathogen of any kind, long or short term.