Viral entry, target cell tropism, and virus receptor interactions
Human retrovirus transmission requires parenteral or intimate mucous membrane exposure to the virus. Transmission by fomites is unlikely because the viral envelope of retroviruses is easily damaged in the environment by exposure to high temperatures, detergents, and chemical disinfectants and through drying. The concentration of virus in body fluids to which a person is exposed is a very important determinant of transmis-sion.17 For HIV, the blood level of virus may or may not reflect the virus level in genital fluids and breast milk.18-20
Receptors
The cellular receptor for HTLV has not been identified. Only T cells are productively targeted by HTLV, but infection of B cells and other cell types has been detected. Much more is known about the manner by which HIV enters the host cell. Susceptible cells must be available for the process of adsorption, penetration, and uncoating of the virus to occur. The receptor for HIV is the CD4 molecule, which is present on T cells and defines the subset of CD4+ helper-inducer T cells. The HIV-1 gp120env protein binds specifically to the CD4 receptor; in ad-dition, other cell surface proteins, such as the chemokine receptors, serve as essential coreceptors for different strains of HIV.
![Illustrated is the replication cycle of HIV-1. The virus must first attach to the CD4 receptor and chemokine coreceptor on the cell surface [see Figure 2]. After fusion of the viral envelope with the plasma membrane of the target cell, the nucleocapsid undergoes uncoating, which is facilitated by the presence of cyclophilin A. The viral RNA genome is reverse-transcribed to double-stranded viral DNA (vDNA), which enters the nucleus as a preintegration complex that contains vpr protein and integrase. Only linear vDNA is capable of integrating randomly into the host chromosome; other forms of partially transcribed linear vDNA fragments and 1-long-terminal repeat (LTR) and 2-LTR circularized, episomal vDNA are not capable of integration. The integrated linear vDNA (now termed the provirus) serves as the template for viral transcription. Transcription of the proviral DNA template yields genomic viral RNA, and alternative messenger RNA (mRNA) splicing creates spliced viral mRNA species that encode the viral accessory proteins and the unspliced viral mRNA species that encode the viral structural polyproteins [see Table 2]. All of the transcripts are exported to the cytoplasm, where translation processing and assembly begins to occur in the endoplasmic reticulum and Golgi complex. The viral polypeptides, protease, viral RNA, and other constituents of the viral core condense at areas of the plasma membrane that have already accumulated viral envelope proteins (gp120/gp41). Budding of the virion ensues, and the immature virion nucleocapsid core undergoes further proteolytic maturation in the extracellular milieu.](http://what-when-how.com/wp-content/uploads/2012/04/tmp10816.jpg "Illustrated is the replication cycle of HIV-1. The virus must first attach to the CD4 receptor and chemokine coreceptor on the cell surface [see Figure 2]. After fusion of the viral envelope with the plasma membrane of the target cell, the nucleocapsid undergoes uncoating, which is facilitated by the presence of cyclophilin A. The viral RNA genome is reverse-transcribed to double-stranded viral DNA (vDNA), which enters the nucleus as a preintegration complex that contains vpr protein and integrase. Only linear vDNA is capable of integrating randomly into the host chromosome; other forms of partially transcribed linear vDNA fragments and 1-long-terminal repeat (LTR) and 2-LTR circularized, episomal vDNA are not capable of integration. The integrated linear vDNA (now termed the provirus) serves as the template for viral transcription. Transcription of the proviral DNA template yields genomic viral RNA, and alternative messenger RNA (mRNA) splicing creates spliced viral mRNA species that encode the viral accessory proteins and the unspliced viral mRNA species that encode the viral structural polyproteins [see Table 2]. All of the transcripts are exported to the cytoplasm, where translation processing and assembly begins to occur in the endoplasmic reticulum and Golgi complex. The viral polypeptides, protease, viral RNA, and other constituents of the viral core condense at areas of the plasma membrane that have already accumulated viral envelope proteins (gp120/gp41). Budding of the virion ensues, and the immature virion nucleocapsid core undergoes further proteolytic maturation in the extracellular milieu.")
Figure 3 Illustrated is the replication cycle of HIV-1. The virus must first attach to the CD4 receptor and chemokine coreceptor on the cell surface [see Figure 2]. After fusion of the viral envelope with the plasma membrane of the target cell, the nucleocapsid undergoes uncoating, which is facilitated by the presence of cyclophilin A. The viral RNA genome is reverse-transcribed to double-stranded viral DNA (vDNA), which enters the nucleus as a preintegration complex that contains vpr protein and integrase. Only linear vDNA is capable of integrating randomly into the host chromosome; other forms of partially transcribed linear vDNA fragments and 1-long-terminal repeat (LTR) and 2-LTR circularized, episomal vDNA are not capable of integration. The integrated linear vDNA (now termed the provirus) serves as the template for viral transcription. Transcription of the proviral DNA template yields genomic viral RNA, and alternative messenger RNA (mRNA) splicing creates spliced viral mRNA species that encode the viral accessory proteins and the unspliced viral mRNA species that encode the viral structural polyproteins [see Table 2]. All of the transcripts are exported to the cytoplasm, where translation processing and assembly begins to occur in the endoplasmic reticulum and Golgi complex. The viral polypeptides, protease, viral RNA, and other constituents of the viral core condense at areas of the plasma membrane that have already accumulated viral envelope proteins (gp120/gp41). Budding of the virion ensues, and the immature virion nucleocapsid core undergoes further proteolytic maturation in the extracellular milieu.
Coreceptors
The HIV-1 coreceptors belong to the superfamily of protein-coupled receptors that bind guanosine triphosphate (GTP). These receptors have a characteristic structure of seven trans-membrane segments and are coupled with the G protein.21 The two principal HIV-1 coreceptors, CC chemokine receptor-5 (CCR5) and CXC chemokine receptor-4 (CXCR4), and their chemokine ligands are involved in diverse biologic processes, such as immunomodulation, inflammation, hematopoiesis, and organogenesis.22 Three cytokines released by CD8+ T cells— RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1a (MIP-1a), and MIP-1P—bind to the CCR5 receptor and may suppress HIV macrophage tropic virus. Similarly, stromal cell-derived fac-tor-1a (SDF-1a), which is the natural ligand for CXCR4, and AMD-3100, which is a small molecule inhibitor of SDF-1a, can inhibit R4 virus.23,24 Clinical development of AMD-3100 was abandoned in May 2001 because of possible cardiac toxicity and limited efficacy. It has been proposed that aberrant signaling through binding of gp120env with the chemokine receptors may cause apoptosis and contribute to the decrease in the CD4+ T cell count in patients with AIDS.
Infection with strains of virus that recognize the CXCR4 coreceptor generally results in cell fusion or syncytium formation in vitro; such strains are termed syncytium-inducing (SI), T-tropic, or R4 strains. Strains of virus that recognize the CCR5 coreceptor generally result in infection without syncytium formation in vitro; such strains are termed non-syncytium-induc-ing (NSI), monocytotropic, or R5 strains. Dual-tropic strains are able to use both chemokine coreceptors. Other chemokine receptors have also been shown to facilitate HIV infection of susceptible cells in vitro.26 Thus, chemokine coreceptor use may be a determinant of viral virulence and disease progression.27 For example, a 32-base-pair deletion in the CCR5 gene, which is found as a homozygous mutation in less than 1% of the Western European white population, prevents surface expression of the truncated CCR5 receptor protein. This deletion is associated with slower progression of HIV-1 infection and a decrease in HIV-1 infection of peripheral mononuclear blood cells in vitro.28 Even heterozygosity for this gene defect, which is observed in 20% of Western European whites, may provide some degree of protection against disease progression.
HIV-1 gp120env also binds to a dendritic cell-specific C-type lectin, DC-SIGN (dendritic cell-specific intracellular adhesion molecule-grabbing nonintegrin), which is highly expressed on dendritic cells in mucosal tissues.31 DC-SIGN does not function as a receptor per se for viral entry into dendritic cells; rather, it promotes infection of T cells that express CD4 and chemokine receptors.32 As such, DC-SIGN efficiently captures HIV-1 in the periphery, stabilizes the virus, and facilitates its transport to secondary lymphoid organs rich in CD4+ T cell targets.
HIV infection is associated with a loss of CD4 antigen on the infected T cell and ultimately leads to the death of the infected T cell. This in turn contributes to a decrease in the ratio of CD4+ T cells to CD8+ T cells—a hallmark of HIV-induced immunosup-pression. However, the CD4+ T cell is not the only type of cell subject to infection by HIV. Other cells, such as the monocyte-macrophage, express CD4 molecules and probably disseminate HIV to target organs. In addition, monocyte-macrophages serve as a reservoir for HIV and other lentiviruses. Other cells can be infected with HIV in vitro, including Langerhans cells of the skin, follicular dendritic cells, and several others, some of which do not express the CD4 antigen. For example, astrocytes, oligo-dendrocytes, M cells of the enteric mucosa, and epithelial cells of the intestine and vagina all express galactosylceramide or a related glycolipid receptor that may act as an alternative virus receptor for gp120env.
Latency and persistence
For any virus infection to persist, viral gene expression must be restricted to some extent. Three patterns of restricted viral expression are known; all three patterns are important for retrovi-ral infections.35 The first pattern, latent infection, is characterized by intermittent episodes of acute or subclinical disease; in latent infection, no virus is detectable between episodes. An example of latent infection is herpes simplex virus infection, which is characterized by episodic subclinical shedding. Latent infection may be particularly important for patients infected with HTLV or HIV-1 whose plasma levels of viral RNA have been suppressed below detectable levels through use of antiretroviral therapy. Latent infection must be differentiated from clinical latency, which occurs in the presence of ongoing viral replication.
The second pattern of restricted viral expression is chronic infection. In chronic infection, the virus is usually demonstrable but disease is absent. An example of chronic infection is cy-tomegalovirus infection. The chronic pattern of infection is seen in approximately one half of the patients infected with HIV-1 who are treated with antiretroviral drugs. In these patients, the plasma levels of HIV-1 RNA are persistently low as a result of potent antiretroviral therapy (HIV-1 RNA counts are greater than 50 RNA copies/ml of plasma but are less than 5,000 copies/ml). However, unlike untreated patients, these patients experience a sustained increase in CD4+ T cells and improved clinical status.
The third pattern of restricted viral expression is persistent infection. This pattern is characterized by a long incubation period with slowly increasing amounts of virus, eventually leading to symptomatic disease. Examples are subacute sclerosing panencephalitis associated with measles virus infection and hepatitis C virus (HCV) infection. This pattern is often seen in HTLV infection, which is characterized by a low probability of developing clinical disease over the infected person’s lifetime (the probability is from 5% to 10%) and a period lasting decades in which the patient is asymptomatic. This pattern is also seen in patients with HIV-1 infection; in these patients, the median time to the development of AIDS is approximately 10 years.
Infections with the lentiviruses, particularly HIV, have features characteristic of all three patterns but are best described as persistent and cytopathic. HTLV infections have the characteristic features of latent and persistent infections, but in addition, the infected cell may be transformed.
Both the HIV and HTLV cDNA become integrated with the genome of the infected host cell at a single random site to establish the provirus as a permanent chromosomal resident.4 In addition, most of the HIV cDNA remains in an extrachromosomal state, either as linear cDNA fragments or as circularized forms (neither of which are replicatively competent). The degree of viral transcription and translation depends on the stage of differentiation of the infected CD4+ T cell. That stage, in turn, is probably related to exogenous antigen stimulation and the action of cytokines (e.g., IL-2) and viral regulatory proteins (e.g., the vprprotein), which promote cell differentiation and activation of viral promotors (e.g., nuclear factor-KB [NF-kB] inducible transcription factors). Integration of human retroviruses differs from that of certain animal retroviruses in that no transforming genes (oncogenes) are associated with the viral genome, and the proviral genome is not regularly inserted next to a host-transforming gene. Moreover, clinically important human retro-viruses are exogenous and are not transmitted in germ cells, as are some endogenous vertebrate retroviruses and the clinically unimportant HERVs [see Table I].
Cytopathicity
The cytopathic destruction of lymphoid cells is a characteristic of lentivirus infection in general. In a minority of patients with HIV, an initial period of intense viral replication is followed by an acute and rapidly progressive disease; in most patients with HIV, however, this initial period of viral replication is followed by a period of persistent infection, with clinical disease developing only after a prolonged period. In some patients in whom disease progression is very slow, chronic infection can develop, and the development of such chronic infection is a viable long-term clinical objective of antiretroviral therapy.
The clinical manifestations of HIV are governed by cell and tissue tropism; the clinical signs and symptoms of infection arise directly or indirectly from viral replication within these cells and tissues. The sine qua non of HIV infection is CD4+ T cell depletion, immunosuppression, and the development of opportunistic infections and malignancies. This depletion of CD4+ T cells occurs not only by direct viral replication and cell lysis but also through other mechanisms. For example, HIV env-induced cell fusion may result in syncytia formation; cell surface expression of the Fas gene and programmed cell death (apoptosis); aberrant signaling through chemokine receptors; arrest of proliferation at the G2 phase of the cell cycle, with cytopathic ballooning of the cells; and antibody-dependent or cell-mediated depletion induced by the binding of gp120env. In addition, the functional responses of T cells, such as signaling, are impaired both in vitro and in vivo because of the binding of gp120env with the CD4 molecule.
A number of other cellular and lymphoid abnormalities are observed in persons infected with HIV, including abnormal B cell immunity and humoral immunity, monocyte-macrophage dysfunction, and immune defects in natural killer cells, among many observed immunologic defects.36 Moreover, the follicular dendritic cells (FDCs) may play a key role in maintaining a large reservoir of protected infectious virus particles on the dendritic cell surface; although these cells do not appear to be infected, the presence of virus particles on the cell surface results in chronic immune activation and cytokine secretion, which leads to further virus replication and dissemination. Eventually, the FDCs are destroyed, which in turn leads to the destruction of the lymphoid architecture and the evolution of clinical dis-ease.36 In many instances, lymphoid architecture may be partially restored after prolonged antiretroviral therapy.
Genomic diversity
HIV undergoes active and continuous replication in the infected person, with infected cells dying after approximately 2 days.37,38 The level of plasma HIV particles remains relatively constant for each infected person because of the balance between rapid production and clearance of virions. To maintain this steady-state level of HIV RNA, there are an estimated one billion infectious events every day in the untreated person with HIV infection. As such, the large number of replication cycles— estimated to be 300 cycles or more a year—provide ample opportunity for the virus to develop genetic diversity as a primary defence against immune and antiretroviral therapy suppression of replication. For example, within the env gene, localized regions of extraordinary hypervariability are interspersed with well-conserved (i.e., nonvariable) regions. One such hypervari-able region is the V3 loop, which is a target for neutralizing antibodies. The force driving this variability is probably caused by selective pressure from the host’s immune system.
This variability within HIV env gene contrasts with the relative genetic stability of HTLV. The mutation rate in the HIV env gene is one million times greater than that of DNA viruses and 10 to 100 times greater than that of other retroviruses; this results in a production of so-called quasispecies and is comparable only to the variability noted for another RNA virus, HCV. This extraordinary env gene hypervariability is associated with amino acid changes of as much as 35% or more among divergent HIV-1 isolates. Variation arises during reverse transcription of viral RNA to cDNA, a step that is highly prone to error, that lacks proofreading (i.e., correction of transcriptional copying errors) because the RNA template is destroyed by RNase H, and that leads to substitutions and misreading, which may result in the modification of several biologic properties, including tissue tropism, virulence, replication rate, and susceptibility to antiretroviral agents. In addition, recombination between different subtypes has been shown to be an important mechanism for generating diversity. Ultimately, continued genetic diversity of HIV may allow the virus to evade containment by the immune system.
Origins of Human Retroviruses
The extent to which different human and primate retrovirus-es are related can be determined by comparing nucleic acid sequences through a process called phylogenetic analysis. Through such analysis, the human retroviruses have been shown to be phylogenetically related to the retroviruses of Old World primates. The closest viral relative of HTLV-I is the simian T cell lymphotropic virus type I (STLV-I), which was identified as an agent of naturally occurring infection in many species of Old World monkeys and great apes. Antecedent infection of the human population probably occurred from three geographically distinct interspecies transmission events; once established, the virus followed the migratory patterns of people around the world.40 Moreover, recent studies have convincingly shown that there have been three separate interspecies transfers of the lentivirus simian immunodeficiency virus (SIVcpz) from chimpanzees (Pan troglodytes) to humans; these transmission events occurred within the past century and resulted in the establishment of HIV-1 groups M, N, and O.4142 Similarly, HIV-2 has its origin with multiple interspecies transfers of SIVsmm from the primate sooty mangaby (Cerocebus atys) to man and to primates in captivity (e.g., SIVmac in the rhesus monkey Macaca mulatta).
Human T Cell Lymphotropic Viruses
Retroviruses cause many animal malignancies and characteristically immortalize cells in vitro. In humans, the prototype retroviruses associated with malignant transformation are HTLV-I (and possibly HTLV-II). HTLV-I causes adult T cell leukemia (ATL); direct infection of the central nervous system results in a spastic or ataxic myelopathy termed tropical spastic paraparesis, also referred to as HTLV-I-associated myelopathy (HAM). HTLV-II was first isolated from a patient with a T cell variant of hairy-cell leukemia; it may be associated with certain neurologic, hematologic, and dermatologic diseases, but unequivocal evidence that HTLV-II is the etiologic agent is lacking.
HTLV-I and HTLV-II have similar genomic organizations and share approximately 60% nucleotide homology45 [see Figure 2]. The gag gene encodes for the structural proteins p19, p24, and p15. The pol gene encodes for the protease and the reverse transcriptase. The env gene encodes for the external envelope and transmembrane glycoproteins gp46 and gp21. The spliced regulatory proteins tax and rex and the other three open reading frames constitute the 9 kb genome. The gag and env proteins are most immunogenic; the antibodies to these proteins are commonly detected by enzyme immunoassay (EIA) and Western blot (WB) assay.
Epidemiology
HTLV-1 has a worldwide distribution; prevalence rates range from 5% to 27%, with the higher rates occurring in certain populations in which HTLV-1 is highly endemic.46 HTLV-II is endemic in several Native American populations in the Americas and in Pygmy tribes in central Africa; in these populations, prevalence rates range from 7% to 9%.44 In the United States, the seroprevalence rates of HTLV-I and HTLV-II range from 7% to 49% in injection drug users and prostitutes.46 Early serologic and epidemiologic studies are confusing, because these studies were unable to effectively differentiate between HTLV-I and HTLV-II; however, this problem has been corrected through the use of more specific recombinant peptide-based WB and nucleic acid amplification methods.47
Classification of htlv subtypes
The HTLV genome is highly conserved, but greater nu-cleotide convergence in the LTRs has made possible the development of restriction fragment length polymorphism (RFLP) testing. Through such testing, it is possible to classify both HTLV-I and HTLV-II into genotypic subtypes, and valuable information on viral transmission has become available.47 There are five major molecular and geographic subtypes of HTLV-I: cosmopolitan (worldwide), Japanese, West African, Central African, and Melanesian.48 There are three HTLV-II subtypes that reflect population clustering rather than geographic clustering: IIa, IIb, and IIc. Subtype IIa is found in injection drug users worldwide; subtype IIb is found primarily in Native Americans; and subtype IIc is found in Brazilian tribes.49
Overview of laboratory diagnosis of htlv
Screening of blood donors for HTLV-I and HTLV-II is now routinely used in the United States, Canada, several Caribbean countries, Europe, and Japan.47 The primary screening assay is the EIA; WB assay is used for confirmatory testing. However, EIA cannot distinguish between HTLV-I and HTLV-II infections because of the significant protein homology between the two viruses. Importantly, antibodies to HTLV do not cross-react with HIV proteins. After repeatedly positive results on EIA, the diagnosis of HTLV infection is confirmed if antibodies to two gene products (gag and env proteins) are detected on WB assay.
For example, a specimen demonstrating antibody reactivity to p24gag and to pg46env or gp61/68env, or both, is considered to be positive for HTLV-I or HTLV-II. For specimens that test positive on EIA and that react with any of the WB bands but that do not meet this criterion, results are considered indeterminate. Specimens that test positive on EIA but that display no im-munoreactivity to any of the HTLV WB bands are considered to be negative for antibodies to HTLV-I and HTLV-II, and the result of the EIA is considered to be false positive. To better differentiate between HTLV-I and HTLV-II, the WB assay has been modified to contain type-specific recombinant proteins from the external glycoprotein of HTVL-I (rgp46envI) and HTLV-II (rgp46envII) as well as a truncated recombinant peptide, rp21e, from the transmembrane glycoprotein gp21env. In one study, persons with indeterminate WB assay profiles (i.e., persons who did not demonstrate antibody reactivity to p24, p19, or rp21e) who did not have risk factors for HTLV infection were shown by polymerase chain reaction amplification not to be infected with either HTLV-I or HTLV-II.50 Such indeterminate WB assay results appear to represent antibodies to other viral and cellular antigens that cross-react with HTLV proteins. Nevertheless, blood donors with indeterminate results are deferred and their blood is excluded for transfusion purposes.
PCR has become the reference method for determining infectious status; for validating serologic assays; for distinguishing between HTLV-I and HTLV-II; for studying in vivo viral load and tissue distribution; and for further evaluating patients with risk factors for HTLV infection whose serologic status is either indeterminate or negative.47 PCR is also an important method of testing infants for HTLV infection, because their serologic status may be unclear, owing to the presence of passively transferred maternal antibodies. In addition, PCR is important for the detection of infection in the window period between exposure, infection, and seroconversion.47
HTLV-I
HTLV-I is the etiologic agent of adult T cell leukemia/lym-phoma (ATL) and a chronic progressive inflammatory neurologic degenerative disorder known as tropical spastic parapare-sis or, more commonly, HTLV-I-associated myelopathy (HAM). However, this virus has been implicated in several other disorders, including an inflammatory arthropathy, uveitis, poly-myositis, infectious dermatitis in children, pulmonary disorders, and Sjogren syndrome. There are provocative but un-proven associations of HTLV-I with mycosis fungoides and Sezary syndrome. This discussion focuses on ATL and HAM because of the established etiologic linkage to HTLV-I.
