Human retroviruses and the acquired immunodeficiency syndrome

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Human retroviruses and the acquired immunodeficiency syndrome I. Virology

update

Maria C. Soto-Aguilar,

MD, and Richard D. desharo,

During the past 5 years, a new form of AIDS has become endemic in Central Africa and common in certain groups of the population in the United States, Europe, and elsewhere. By 1983, many of the immunologic abnormalities, infections, and malignancies, which are now commonly associated with AIDS, had been reported; however, the etiology remained an enigma. ’ Because of the rapid spread of AIDS, a remarkable series of investigations have been performed in a very short time. After intensive basic and clinical research carried out in the United States and France, the causative agent of AIDS and related diseases was identified as a T-lymphotropic retrovirus.* This virus, called HTLV-III, LAV, or the HIV, has unique properties

From the Clinical Immunology Section, Departments of Medicine and Pediatrics, Tulane University Medical Center, New Orleans, and the Tulane/Louisiana State University AIDS Treatment Evaluation Unit (ATEU publication No. 005-M), New Orleans, La. Supported by National Institutes of Health Grant AI-72625. Received for publication Jan. 1, 1987. Accepted for publication Aug. 11, 1987. Reprint requests: Maria C. Soto-Aguilar, MD, Tulane University, Dept. of Medicine, Room 7209, 1430Tulane Ave., New Orleans, LA 70112.

MD New Orleans. La

Abbreviations used AIDS:

Acquired immunodeliciency syndrome

HIV-l:

Human immunodeficiency virus, formerly HTLV-III/

LAV

HTLV: LAV: RNP: LTR: PV: mRNA: ARV: STLV-IIImac:

Human T-lymphotropic virus Lymphadenopathy-associatedvirus Ribonucleoprotein Long terminal repeats Proviral Messenger RNA AIDS-related virus Simian T-lymphotropic virus type III

STLV-IIIagm:

found in rhesus macaques Virus found in green monkeys

RIPA: Radioimmunoprecipitation analysis

that result in genetic modifications of host cells, leading to dysfunction and/or premature death. Present research is focused on the mechanisms causing immunologic and neurologic injury and mechanisms that modulate the clinical manifestations of infection that range from apparent health to full-blown AIDS. In this article, we will review available information 619

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-Viral

envelope

Reverse

glycoproteins

transcriptase

RNA Capsid Envelope

FIG. 1. Retroviral

on the virology of HIV and attempt to relate this information to present notions of the origin of AIDS. This information is necessary to understand present attempts at drug therapy of retroviral infections. GENERAL FEATURES OF RETROVIRUSES Viral structure Viruses are obligate cellular parasites too simple in structure to be regarded as microorganisms. They contain only one type of nucleic acid, either RNA or DNA, single stranded or double stranded, and have no ribosomes, mitochondria, or other organelles. Therefore, they are completely dependent on the cellular machinery of their host for the production of energy, synthesis of structural macromolecules, and replication.3 Retroviruses are a family within the RNA viruses with a particular ability to integrate their viral RNA into the host cell DNA using a special viral enzyme called reverse transcriptase. Vii-ion forms of Retroviridae are infectious particles of 100 nm in diameter containing RNA, with an external lipid bilayer derived from a host cell from which loosely attached surface knobs (virus-coded glycoproteins) project. Below the virus envelope, there is an inner coat that surrounds the virus capsid, a shell in the virus core that contains the RNP complex. The RNP consists of a filamental strand of RNA in the form of a spiral containing reverse transcriptase4 (Fig. 1). The viral genetic code or genome of the virus is made up of three types of genes? (1) genes for enzymes that participate in the replication of the viral nucleic acid, (2) genes for proteins that are involved in regulatory processes, and (3) genes coding for the structural viral proteins or proteins needed to form the viral capsids and envelopes. Thus, the genetic composition of retroviruses is RNA in the extracellular particle and PV DNA in the infected ce11.6

virion.

Life cycle of retrovirus There are five stages in the retroviral infection cycle5,’ (Fig. 2). Virus infection at the host-cell membrane. The first stage is attachment of the virus to a host cell through a specific interaction between a receptor on the hostcell membrane and the viral envelope. The next step is penetration and uncoating of the virus, which involves fusion of the virus membrane with the plasma membrane of the infected cell or formation of endocytic vesicles in which the virus is taken inside the cell by endocytosis. Cytoplasmic lysosomes join the endocytic vesicles, and at the acid pH of the lysosome, the envelope of the endocytosed virus fuses with the lysosomal membrane, and the viral nucleocapsid is expelled into the cytoplasm. In the case of the AIDS virus, this process involves interaction between a viral envelope glycoprotein and a protein called T4 receptor of CD4 T-lymphocytes (helper / inducer population). Synthesis of viral DNA in the cytoplasm. Once in the cell cytoplasm, the viral reverse transcriptase (RNA-dependent DNA polymerase enzyme) transcribes the viral RNA into single-stranded DNA (complimentary DNA, cDNA). This cDNA is copied by the same enzyme into double-stranded DNA, which encodes the viral genome and is called “PV DNA.” This PV DNA has infectious capacity,6 but like other virus DNAs, the provirus is a replicative intermediate that requires integration into host-cell chromosomes to be able to replicate itself.* Linear forms of doublestranded DNA change to circular provirus forms, presumably by cellular enzymes like ligase4 that seal the 3’ and 5’ ends of the DNA fragments called LTRs. LTRs are sequences of nucleotide bases with an identical region in common and serve to direct RNA transcription into DNA from 5’ to 3’ ends. This allows formation of circular provirus forms. Integration of PV DNA into the cellular DNA in the

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nucleus. The PV DNA moves from the cytoplasm to the nucleus and becomes stably integrated into the host genome (cellular DNA). Integrated viral genes then are duplicated with the normal cellular genes and are indistinguishable from any other cellular gene. Some viral double-stranded DNA genomes are retained in the nuclei of infected cells as episomes, whereas others are transcribed into RNA and mRNA with host DNA-dependent RNA polymerase. This leads to the production of new virions. Virion assembly in the cytoplasm. Newly produced viral mRNA goes to ribosomes in the cytoplasm, and viral structural proteins are synthesized, assembling viral nucleocapsids .5 Budding of virions at the cell sueace. The viral proteins are probably transported to the cell surface, where packaging of polypeptides and RNA and subsequent budding of virions from the cell membrane take place. New viral particles are released to the extracellular space, and these particles can infect other susceptible cells (Fig. 2). Cellular

consequences

of viral infection

The integrated viral DNA may lead to changes in the expression of nearby cellular genes, which may result in neoplastic transformation of the cell, premature degeneration, and early death, called terminal differentiation, or cytopathic effects and dysfunction.’ If proteins in the viral envelope cross-react with normal cellular proteins, autoantibodies or immune complexes may form, and autoimmune disease (such as idiopathic thrombocytopenic purpura in patients with AIDS) may occur. The viral envelope proteins, themselves, may be immunosuppressive.‘, ‘O Endogenous

and exogenous

retroviruses

According to their mode of transmission, retroviruses can be categorized as endogenous or exogenous.’ Endogenous viruses are not known to cause disease in nature. They are transmitted in the germ cell line along with normal genetic elements, not altering the cell function. Such PV DNA is found in the DNA of all normal cells of many species of animals and, under certain circumstances, may be induced to produce 1, VlruS . Exogenous retroviruses cause disease in most animals and humans, acting as infectious agents. They can induce neoplastic changes in the infected host or induce

Human

nononcogenic,

cytopathic

damage.

T cell retroviruses

In 1978, the first isolates of human retroviruses were isolated” at the National Cancer Institute, Bethesda, Md., from patients with T cell lymphopro-

FIG. 2. Life cycle of retroviruses. (1) An RNA virus particle (A) fuses with a cell (BJ, and the RNP of the virus becomes uncoated, expelled into the cytoplasm (CJ. (2) The viral reverse transcriptase (RNA-dependent DNA polymerase) transcribes the viral RNA into single-stranded DNA (AI -+ synthesis of RNA-DNA hybrid, followed by synthesis of double-stranded DNA (B). Linear progeny double-stranded DNA -+ circular double-stranded DNA or pravirus (C) by sealing of 3’ and 5’ terminal segments of the PV genome. (3) Circular double-stranded DNA enters the host cell nudeus (A) and integrates into cellular DNA (B), Viral DNA in the chromosomal DNA + cellular DNA-dependent RNA polymerase II of the host cell + viral mRNA (C) that goes to ribosomes in the cytoplasm. (4) Viral mRNA plus ribosomes --+ viral proteins (A). Viral proteins (VP) plus RNA molecules + viral nucleocapsids (B). (5) Nuclaocapsid.s plus cellular membranes (containing viral glycoproteins) (A) -+ budding of virions (B) and release into the extracellular space (Cc).

liferative disease. These viruses could infect and transform T cells in culture and were named ‘*human T cell leukemia/ lymphoma virus” (subsequently called HTLV-I). Later HTLV-I was found to be endemic in southern Japan, Africa, and southeastern United States. A second human retrovirus was identified in the cells of patients with hairy-cell leukemia, another rare form of cancer, and was designated HTLV-II. Influenced by these discoveries, Gallo. Essex, and other investigators began to think as early as 1982 that a retrovirus variant of HTLV-I could be the agent of AIDS. Instead of causing T cell proliferation. such a virus might be cytotoxic for the cells. In November 1982, these investigators were able to detect reverse transcriptase activity in cell cu!tures from

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TABLE I. Retroviruses”

I. Endogenous II. Exogenous A. Chronic leukemia viruses 1. Mouse leukemia virus 2. Feline leukemia virus 3. Avian leukemia virus

4. Gibbon ape leukemia B. Acutely transforming viruses 1. Avian sarcomavirus 2. Avian myeloblastosisvirus 3. Avian myelocytomatosis virus C. T-lymphotropic retrovimses 1. HTLV-I, HTIY-II, and HTLV-III 2. Bovine leukemia virus

3. Lentivimses *Adaptedfrom Gallo andWong-Staal.’

AIDS patients, when the primary cells were supported with T cell growth factor (interleukin-2). The cells, unfortunately, quickly degenerated, and no virus could be recovered.* At this point, they named the suspected AIDS virus HTLV-III, and the name of the whole family was changed to “human T-lymphotropic viruses.” Montagnier, and other investigators at the Pasteur Institute, were also working on the AIDS problem. In January 1983, they cultured lymph node cells of a biopsy specimen obtained from a patient with lymphadenopathy. The culture was supported with interleukin-2, and by adding fresh lymphocytes periodically, once they observed that the cells rapidly died. Viruses propagated well in this cell culture system, and the first virus was soon isolated. This virus failed to react with monoclonal antibodies to the core proteins of HTLV-I supplied by Gallo. The Pasteur group did some biochemical characterizations of the virus, obtained the first electron micrographs of virus particles budding from the surface of infected cells, and called their isolate LAV The virus could not be grown in quantity. Subsequently, the American group discovered a line of T cells from a leukemia patient (cell line H9) that could be directly infected from the cells of patients with AIDS and produce virus indefinitely. Mikulas Popovic from Gallo’s laboratory pooled virus isolates from 10 patients and used the mixture to infect the cells. HTLV-III virus was then identified, and by December 1983, the virus was being mass produced for the first time. This allowed careful studies of viral antigens and development of diagnostic tests. Controversy between the French and the American groups, regarding credit for virus discovery and patent issues

for serologic assays, developed about this time.13‘16 A third group, headed by Jay Levy of the University of California at San Francisco, isolated another virus from patients and subjects from groups at risk for AIDS, designated ARV.“, I8 This virus turned out to be similar to HTLV-III and LAV, but demonstrated genetic differences that placed it as a separate isolate. HTLV-I and HTLV-II retroviruses are oncogenic and appear to be genetically and functionally similar to the STLV-I (probably of African origin) and to the bovine leukemia virus. All these viruses result in malignant proliferation of infected cells. HTLV-III, in contrast, together with LAV and ARV, have a cytopathic effect on human T-lymphocytes, leading to immunodeficiency. Human immunodeficiency virus The retrovirus implicated as causing AIDS has been designated different names by the different groups of investigators, although all share biologic, morphologic, and immunologic properties and have demonstrated to be highly related at a nucleic acid level. To deal with differences in nomenclature, the Executive Committee of the International Committee on Taxonomy of Viruses’9~‘Oagreed that all the isolates represent the same virus, and in May 1986, proposed its official designation as the “human immunodeficiency viruses,” to be known as HIV. The new name describes the host, indicates the major biologic property of the virus, and implies an independent virus species distinct from the names of other retroviruses, and therefore, it also avoids any controversy regarding priority of discovery of the virus. Subsequently, HIV will be used as synonymous with HTLV-III and LAV. Genetic

structure

of retroviruses

Retroviruses have variations in their genomic structure that result in differences in viral proteins. These differences may be of considerable importance in the immunologic responses generated. Gallo and Wong-Staalg have classified the retroviruses in three categories according to the viral genome (Table I). Chronic leukemia viruses. Chronic leukemia viruses are the commonest retroviruses in nature and include mouse leukemia virus, feline leukemia virus, avian leukemia virus, and gibbon ape leukemia virus. They require a long latency period for viral integration on a specific chromosome. Repeated infections appear to be required to induce persistent infection. These viruses contain three viral genes for replication: gag (group-specific antigen gene) for the viral internal structural proteins, such as the viral core proteins, pol (polymerase gene) for the production of reverse tran-

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yt

Viral Genolae

Viral Products

Ccne

Loc.tlon viral in the

of Gene Virus

Products

FIG. 3. Human

immunodeficiency

scriptase, and env gene for the envelope. As is the case with other retroviral genes, when they are integrated into the DNA, the genes have regulatory elements on each end of their genetic sequence called LTR sequences: 5’ LTR -

.gag

PO1

*-LTR3’ env

The LTR sequences act as transcription initiation signals and regulate the expression of viral genes. They “flank” viral genes within the host-cell DNA (called provirus) and also regulate the expression of nearby cellular genes when the virus is integrated. Acutely transforming viruses. These viruses are rare in nature and include avian sarcoma viruses, avian myeloblastosis virus, and avian myelocytomatosis viNS . No viruses of this type have been found in humans so far. Acutely transforming viruses have an extra gene (oncogene) with the special capacity to react with a host gene (protooncogene) in such a way that a malignant gene product results. The result of this interaction (recombinational event) is the insertion of a cellular gene into the viral genome (cell-derived oncogene) so that, on future infection of animals of the same species, the retrovirus can lead to a rapidly developing cancer within days or weeks.4 T-lymphotropic retroviruses. This group includes all the known human retroviruses, bovine leukemia virus, and lentiviruses. These viruses contain one or more extra genes, not derived from infected cells, in addition to the three standard viral genes. HTLV-I,

virus

(HIVIHTLV-III.:LAV).

HTLV-II, and bovine leukemia virus contain an extra gene located between the env gene and 3’ LTR sequence, initially called pX (for the region of undefined function), then lor (for long open reading frame), and later tat (for transacting transcriptional regulation). This gene encodes a nuclear protein that enhances the transcriptional activity of the viral LTR, critical for virus replication. It is believed that the tat protein interacts also with specific cellular-regulating elements, such as genes that are present on lymphoidderived cells (specific for immunoregulatory functions) and that it might be able to regulate their function. The seven known immunodeficiency

genes of the human virus

HIV contains the three genes (called open reading frames) of other retroviruses genome (gag, env, and pal) that encode, respectively, the capsid proteins, the envelope proteins, and nonstructural proteins necessary for replication. However, its genome also contains other open reading frames that appear to encode several other proteins, not common to mOSt retroviruses*‘.‘* (Fig. 3). Starting at the amino terminus after the 5’ LTR sequence is the gag gene, which encodes a 55,000dalton protein present in large amounts in virusinfected cells, This nonglycosylated protein is the precursor of three capsid or core-related pmteiAS. A protease encoded by the viral pol gene cleaves the 55kd protein and forms these three structural gag-derived

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FIG. 4. Specific antibodies to HTLV-III in a patient with primary HTLV-III infection, as detected by Western blot (pane/ A) and RIPA (panel B). Lane a illustrates 30 weeks before illness; lane b, during illness; lane c, 2 weeks after illness; and lane d, 11 weeks after illness. (From Ho DD, Sarngadharan MG, Resnick L, et al. Primary human T-lymphotropic virus type Ill infection. Ann Intern Med 1985;103:880. By permission.1

proteins, p17 (18), p24 (25) and ~13, a peptide of about 13 kd. Peptides p24 and p17 are detectable in extracellular virus and disrupted virus-infected cells, but the 55kd protein is not present in significant amounts in the virus itself. The second virus gene known is pal, the most stable (conserved) gene in retroviruses. It encodes for the reverse transcriptase and endonuclease activities of the virus. Next to it is a third gene called sor for “short open reading frame” whose product is a 23-kd protein of unidentified function. The fourth gene is the transactivator tat-Ill, a gene with two components (bipartite gene), consisting of one functional set of nucleotide bases (exon) located between the sor and env (envelope) genes, and another exon located within the env gene in a different reading frame. Recently, Fisher et al.,23 using genetically modified HTLV-III viral DNA (plasmids) that lack tat-M, were able to demonstrate that tat-ZIZ is essential for virus expression. Tat-III encodes a 14-kd protein that stimulates the expression of the genes linked to HIV LTR sequences, after the transcription of PV DNA into RNA has occurred (posttranscriptional event). Therefore, it stimulates viral replication in infected cells. The fifth gene, known as env is a large open reading frame that encodes for a polypeptide in the range of

110,000 to 120,000 molecular weight. This protein has numerous glycosylation sites and is the external envelope glycoprotein of HIV. It is present in infected cells only and migrates in gel electrophoresis as a 150to 160-kd glycoprotein (gp150). It is processed into a second precursor of 135 kd, and, finally, through proteolytic cleavage and carbohydrate modifications, into the virion glycoprotein called gpl lo-gp120, which serves as envelope attachment unit or peplomer, and into the glycoprotein gp41-gp42, a transmembrane protein*’ (Fig. 3). A sixth gene designated 3’orf, located in the 3’ region of the HIV genome, has been found to encode for a 27-kd protein (~27) that can be recognized by the immune system and elicit antibody response in infected individuals. 24 Finally, a seventh viral gene necessary for replication has been recently described by Sodroski et al.” designated art (antirepression translation). It is a second bipartite transactivator gene whose 116aminoacid product activates the expression of the gag and env HIV genes and thus also regulates virus replication. 25, *’ These investigators postulate that art relieves or counteracts negative regulatory sequences present on viral RNA messages that inhibit the production of capsid and envelope structural proteins.

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Oncovirinae

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Lentivirinae

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syr~rom~!

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(pathogenic) ---

Lymphotropic

.Other FIG. 5. The family of T-lymphotropic retroviruses. merly HTLV-IIIILAV; STLW, simian T-lymphotropic virus, formerly simian T-lymphotropic virus type virus type IV; HIV-Z, human immunodeficiency associated virus type II (LAV-2); V, visna virus equine infectious anemia virus.

Regulation of expression gene products

of viral

The expression of HIV structural genes is governed posttranscriptionally by the fat-Ill gene product that acts as a positive regulatory factor, and by the art product that apparently counteracts negativeregulatory sequences located in or near the gag and env genes. The important functional implications of these regulatory proteins are that the activity of fat-M and art may be essential for the length of HIV latency period, T cell activation, cell killing, or cell transformation. The early stage of infection is characterized by the accumulation of viral RNA but not of virion proteins and, a late stage, is one in which viral proteins toxic to T4 positive T-lymphocytes are produced. Present data suggest a postinfection switch in the production of HIV gene products regulated by activation of either one or both of the rut-ZZZand art genes. The switch triggers a burst of expression of gag and env gene products, and virus production is initiated. 26 lmmunogenicity of viral proteins Some viral proteins are more immunogenic than others. As with other viruses, the surface or envelope proteins (encoded by the env gene in HIV) are more immunogenic in the infected host than unexposed proteins found in the virus core. The env gene proteins gp120 and gpl50 (160) are the most immunogenic, with antibodies present in 97% to 100% of the patients with AIDS or AIDS-related complex.** The third envelope protein, p41, and the core proteins p55 and

Retroviruses?

HIV-l, human immunocleficiency virus, forvirus type I; S/V, simian immunodeficiency Ill (STLV-III); HTL V-/V, human T-lymphotropic virus type 2, formerly lymphadenopathy(sheep); C, caprine encephalitis virus; and f?,

p24 are moderately immunogenic, whereas pl7 (encoded by gag) and p27 (encoded by 3’orj? are weakly immunogenic. lmmunodiagnostic

tests for HIV infection

There are several immunodiagnostic tests available to detect HIV infection. ELISA, the immunoblotting (Western blotting) and radioimmunoprecipitation analysis (RIPA, with sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis) are the most usefu12* ELISA is the most commonly available technique for blood screening, with a sensitivity of 93.4% to 98.9%, and specificity of 99.2% to 99.8%.“’ ELISA elicits a spectrophotometric reading of the amount of antibody binding to HTLV-III antigens from virus grown in the leukemic T cell line H9. False positive ELISA tests have been most frequently found in multiparous women, blood transfusion recipients, and in cases of cross-reactivity, with certain IJLA antigens present in the H9 cell culture (in this setting, antibodies to DR4 have been identified). Western blotting test is a valuable confirmatory assay when antibodies to HIV are detected by ELISA. The Western blot technique uses an electrophoretic process to separate viral antigens and measures reaction of serum antibodies with specific viral protein bands. Its specificity depends on an adequate virus antigen substrate, and a homogenate from infected cells is preferred. In this assay, the two principal protein bands observed are p24 (core polypeptide) and p41 (envelope glycoprotein). The RIPA test, with whole cell homogenates, is the most specific test available because it

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detects antibodies to all the virus antigens and allows the visualization of reactivity to all HIV-specific proteins at one time. The results of Western blot and RIPA prospective analyses performed by Ho et a1.28in a patient with primary HTLV-III infection is illustrated in Fig. 4. SALIENT FEATURES OF HIV Arguments over nomenclature

As noted by Broder and Gallo” and by Essex et al. ,** the three recognized HTLVs share several biologic features, including that (1) they are exogenous viruses that may be isolated from mature T cells, especially T cells identified by the monoclonal antibody OKT4 (CD4+ cells), (2) they infect mature T cells in vitro, (3) they have a reverse transcriptase of similar size and preference for Mg’+ rather than Mn++ as a cofactor, (4) they possess some crossreacting antigens related to membrane or envelope proteins, (5) they have major core proteins of small size (p24/25), (6) they exhibit some homology in nucleotide sequence and have doubly spliced mRNA, (7) they have a transactivating virus gene that regulates transcription (tat), and (8) on in vitro infection of some T cells, they induce formation of multinucleated giant cells. Because of the above characteristics and a hypothesis that the three members of the HTLV family originated in Africa,*’ Broder and Gallo,‘* and Essex et al.‘* prefer to place the AIDS virus in a special category common to all human T-lymphotropic viruses, separate from other retroviruses in nature. White and Fenner,3 Levy et a1.,17 and the Pasteur Institute group,*’ however, believe that the genomic structure of the AIDS virus shares more characteristics with members of lentiviruses (slow viruses), a subfamily in nature of nononcogenic, pathogenic Retroviridae. They believe that HIV is the human equivalent of lentiviruses, which in nature cause disease in sheep (visna and maedi viruses), goats (caprine arthritis and encephalitis virus), and horses (equine infectious anemia virus). Clinical characteristics common to lentivirus and HIV infection include long latency periods, involvement of the hematopoietic and nervous system, and the cylindrical nucleoid structure of the virus particle. Biologic similarities include cytopathic effects in infected cells, such as fusion and multinucleated cells, accumulation of unintegrated circular and linear forms of PV DNA in infected cells, and latent infection in some infected cells. Similar molecular properties include large envelope glycoproteins leading to large provirus size (9.7 kb), the use of lysine transfer-RNA

as an initial binding site for protein synthesis (unlike other retroviruses that use proline transfer-RNA), and a high degree of genetic variation (genetic polymorphism). Genetic

polymorphism

HIV is a highly replicating virus in vivo, with genetic polymorphism not found in other viruses of the HTLV family. This is specially the case in the envelope glycoproteins that appear to be responsible for viral tropism for T4+ lymphocytes and for viral pathogenicity. “, *I Nucleotide (nucleic acid) sequence data of RNA from different viral isolates indicate that HTLV-III and LAV are very similar to each other. With a genome of almost 10,000 nucleotides, LAV and HTLV-III differ by only about 150 nucleotides, whereas ARV differs by almost 600 nucleotides from HTLV-III / LAV. 3o The cloned HTLV-III genome used by Gallo’s group in many studies originated from a cell line infected with virus isolates from 10 different patients and at least four different viruses integrated into the cells in that line. After the discovery of LAV, the Pasteur Institute*’ group isolated other viruses from patients with AIDS and lymphadenopathy, variants of LAV, which they called immune deficiency-associated viruses (IDAV 1 and IDAV2). In addition, they reported finding a distantly related virus, at least 30% different in sequence from HTLV-III/LAV, in two patients with AIDS in a Lisbon hospital.3’, 32The patients’ sera demonstrated no antibodies against LAV; however, the viruses appeared to be like LAV in the electron microscope and had the same T-lymphotropic and cytopathic properties. The newly discovered virus was originally called LAV type II by the investigators, but in a subsequent study of 30 patients from West Africa, they termed it HIV type 2 (HIV-2) and referred to the characterized HIV isolates from North America, Europe, and Central Africa as HIV- 1.33 Other evidence of the genomic heterogeneity of individual AIDS isolates comes from a multicenter study34 that revealed significant nucleotide sequence differences in viral isolates of patients with AIDS from Zaire, as compared with HTLV-III and LAV, whereas individual isolates from New York patients were different from one another, but to a lesser degree. Hahn et aL3’ examined sequential virus isolates from three persistently infected patients with AIDS or risk factors for AIDS. Four to six virus isolates were obtained from each individual during a l- or 2-year period, which differed on isolated and clustered nucleotide point mutations, as well as short deletions or insertions. The comparison of genomic mapping and

VOLUME 13? NLMBER 4

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nucleotide sequence revealed that the rate of genetic evolution is approximately lo-’ to 10m3 nucleotide substitutions per site per year for env and tenfold less (I (I--“) for gag, values that are a millionfold greater than for most DNA genomes. The viruses isolated sequentially from any one patient appear much more related to each other than to viruses from other individuals, suggesting that those viruses have evolved in parallel from a common progenitor virus and that some type of interference mechanism may prevent simultaneous infection by more than one major genotypic form of virus. These results raise the possibility that viral properties, such as tissue tropism, virulence, rate of replication, sensitivity to antiviral drug therapy, and resistance to immunologic attack, may display variation in different individuals. The mechanisms by which genetic variation occurs in HIV are*: 1. Hypervariable regions within the env gene, interspersed with regions of strong conservation 2. Duplications, insertions, deletions, or point mutations of short stretches of nucleotides 3. Errors in reverse transcription leading to nucleotide substitutions and copy-choice misreading of the viral RNA 4. Possible recombination between different viral molecules All these changes result in variations on glycosylation sites of the envelope proteins and therefore lead to modifications in the viral antigenic expression. Origin

and evolution

of the AIDS virus

International epidemiologic studies done on individuals with serologic evidence of HIV infection and ethnic data support the hypothesis that the human retroviruses originated in Central Africa and were distributed to other continents by way of the slave trade. This hypothesis is especially strong in the case of the HTLV-I virus, which is present in Africa, Japan, the United States, and the Caribbean. I2 During the sixteenth century, Portuguese explorers came to southem Japan, and the port of Nagasaki became a large commercial center. Portuguese seamen who had contact with Central Africa, took Africans with them to Japan, and probably monkeys as well, and later came to the Americas with the slave trade. It is probable that they also took the retrovirus with them to these regions and, after the seventeenth century, to Europe.’ Based on the presence of antibodies in healthy individuals and in patients with chronic leukemia/lym-

*Adapted

from

Hahn

et al.”

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phoma, HTLV-1 is known to be widely distributed in Africa. Also, a virus very similar tc! HTW-I called STLV-I occurs, and STLV-I has been t’l)und in man] Old World monkeys from Africa. These viruses are identical to those present in Japanese macaque monkeys. The fact that HTLV-I disease 75 &:ndernic in southern Japan, therefore, had led investigators to think that the virus was either transmltted from infected African individuals or from infected monkeys. and later brought to the western hemisphere.-” The situation with HIV or HTLV-II1 LAV may be similar. Five percent to 10% of the population of African equatorial countries like Zaire and the Central African Republic have been found to hc scropositive and to have a high incidence of aggressive Kaposi’s sarcoma and immunodeficiency. Retrospective serum analysis obtained by French investigators suggests that the HIV virus originated in Africa but has spread to France and other Western countries alncc the early 1970s.” In contrast to their findings oi viral disease association, Saxinger et al? found that rn Uganda (East equatorial Africa), 65% of sera obrained in 1972 from apparently healthy children had antibodies that reacted with HTLV-III. This observation suggests that the antibodies are likely to be the result oi‘ infection with the same or a similar virus. Researchers from the New England PrImate Center discovered a virus similar to HTLV-III in captive rhesus macaques with an AIDS-like dixease, christened STLV-IIImac. The same virus. however, or one very much like it, was found in about 50% of healthy African green monkeys living in the wild (STLV-IIIagm). The STLV-IIIagm viruti has a cytolytic effect on T4 lymphocytes, Mg-’ --dependent reverse transcriptase and retroviral particles with morphology similar to HTLV-III/LAV.” ” it i;i possible that STLV-IIIagm virus might be more immunogenic. and Essex postulates that there may be a spectrum of related viruses that could infect different primate hosts with a range of pathogenic effects ranging from none to full-blown AIDS.” In contrast, the c\ose relation of STLV-IIIagm to HIV raises the possibility that a family of related viruses may have existed in primates for a long time before the AIDS epidemic began and that STLV-III may have been transmitted to humans at some time. AIDS is endemic in Central Africa. and the HIV, like other sexually transmitted agents. appears to infect both male and female subjects, with a higher prevalence in female prost!tutcLb md their contacts. Kanki et al.,” from the Harvard School of Public Health, have reported a 5% incidence of infection in healthy prostitutes from Senegal (West Africa’). Those seropositive individuals displayed strong antibody

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reactivity to all major STLV-IIIagm viral proteins (including the gp120/ 160, ~55, and 24) but demonstrated variable or no reactivity with the major viral antigens of HTLV-III I LAV. The investigators obtained peripheral blood lymphocytes from eight STLV-IIIagm antibody-positive subjects and placed them in coculture with a human mature T cell line. Twenty to 28 days later, they observed atypia and multinucleated giant cells, and at days 28 to 35, viral proteins and budding particles from infected cell membranes were detected. Biochemical analysis and serologic data revealed that this new virus shares more common antigenic sites (epitopes) with STLV-IIIagm than with the prototype HTLV-III/LAV that infects people in the United States and Europe and, for this reason, has been called HTLV-IV.39a a Unlike AIDS-associated viruses (HIV) and LAV-II discovered by Montagnie? (which is also more closely related to STLV-III than to the AIDS virus), HTLV-IV did not kill the cells it infected and did not appear to be associated with any illness. The discoverers of LAV-II (now termed HIV-2), however, believe that HTLV-IV is identical to their isolate because they are antigenically similar and both have been obtained from residents of West Africa. Evidence to date indicates that HTLV-IV may have been present in a small proportion of people in West Africa for more than a decade in the absence of AIDS or a related disorder, but a new epidemic is possible. It is probable that in a near future, antigenic components of HTLV-IV I LAV-II need to be included in diagnostic preparations. It is possible that STLV-IIIagm or HTLV-IV may have served as a progenitor virus to the human AIDS virus and represents a continuum of retroviruses of which LAV-II forms a part40 Coinfection with human T-lymphotropic retroviruses

The prevalence of coinfection with various human T-lymphotropic retroviruses is unknown. One patient with coinfection with HTLV-I and HTLV-III has been reported4’ who developed a lymphoproliferative disease of CD8 + cells. Epidemiologic studies are under way to investigate this problem. Proposed

family

J. ALLERGY CLIN. IMMUNOL. APRIL 1988

and deShazo

tree for retroviruses

With the available information9~ 22x39and with some modifications of a model proposed by Gallo and Wong-Staal,9 the following family tree of transactivating retroviruses could be proposed (Fig. 5). Simian AIDS has been induced in rhesus macaques at the Delta Regional Primate Research Center of Tulane University with inocula from asymptomatic sooty mangabey monkeys. The infected macaques

developed HTLV-III cross-reactive antibodies. The virus isolates obtained appeared to have common immunoreactive epitopes with HTLV-III, such as 120k env and 24k gag-related proteins. Analysis of STLV-III/ Delta virus suggests that the virus is related to STLV-IIImac from the Boston Primate Research Center but is not identical.42 STLV-III/Delta was found to be endemic in the African mangabey colony with no disease association, whereas the Asian (macaque) species acquired the immunodeficiency syndrome. In summary, the presence of HTLV-III-related retroviruses in at least two African primates supports the hypothesis that HIV emerged in humans as a result of an early transspecies transfer in Africa. The difference in susceptibility to disease may be related to different mechanisms of viral recognition by the host, different immunogenicity of the retroviruses, and differences in the inherent pathogenicity of the virus itself. CONCLUSIONS

Our present understanding of the probable origin and cause of AIDS has resulted from the efforts of a large group of international investigators. The following statements summarize present notions: 1. HIV is a highly replicating retrovirus that infects human cells, including T-lymphocytes, particularly those with CD4 surface antigen (OKT4 + or Leu-3 + cells) that comprise the helper/inducer T cell subpopulation. The effects of this lymphotropism include cytopathic changes, giant cell (syncytium) formation, immune system depression, and premature cell death. 2. Nucleotide sequence analysis of HIV virus isolates from different individuals indicate that the AIDS virus consists of a group of related retroviruses that share the same properties of the originally described HTLV-III and LAV but differ by genetic polymorphism, especially in the elzv gene that encodes envelope glycoproteins. For this reason, a new international name has been assigned, and the virus is now called human immunodeficiency virus or HIV (HIV-l). 3. In comparison with human T-lymphotropic viruses type I and II, HIV differs in its replicating capacity, genetic polymorphism, and by having a more complex genome. These differences account for important results, including diversity of antibody production by infected hosts and the broad spectrum of clinical disease produced by infection. 4. Because of these properties and other properties such as long latency period and neurotropism, HIV appears to be the human equivalent of ungulate

VOLUME 81 NCiMRER 4

lentiviruses and, therefore, appears to many investigators to belong in this category of the Retroviridae family tree. HTLV-I and HTLV-II have distinct features that place them within the Oncovirinae group of retroviruses. Epidemiologic studies in man and in monkeys have linked HIV to simian T-lymphotropic retroviruses that cause immunodeficiency syndrome in some species (simian AIDS) and no disease in others. A new type of human T-lymphotropic retrovirus (HTLV-IV/LAV-II/?HIV-2) has been identified in West Africa that shares more antigenic and genetic characteristics with STLV-III than HIV The epidemiologic significance of this finding is uncertain. This suggests a spectrum of virus-related disease and a transspecies transfer from nonhuman primates to man. Historical information and the preceding findings support the hypothesis that human retroviruses originated in Central Africa (where HIV is endemic among human and simian populations) and were transferred first to southern Japan during the sixteenth century and afterward, to America and Europe by way of the slave trade. Although the AIDS virus has existed in Africa and other areas of the world for a prolonged period of time, it has spread only since the early 1970s. Furthermore, it is probable that the virus has become pathogenic through genetic mutations or activations in the past decade. Note added in proof. Since submission of this article. several other authors have concluded that HIV is a retrovirus with special characteristics that include it within the group of the lentiviruses, rather than within the oncoviruses. Ho et al., in their recent article, “Pathogenesis of Infection with Human Immunodeficiency Virus,” N Engl J Med 1987;317:278-86, have a similar hypothesis to ours as to the origin of HIV and the evolutionary relationships among retroviruses. Another major development occurring in the past year has been the introduction of a new serologic assay to detect HIV antigens in blood and other body fluids. This so-called HIV antigen-capture test is a sandwichtype immunoassay (ELISA technique) in which human and rabbit polyclonal anti-HIV IgG are used as capture and probe antibodies, respectively. Then, a second labeled antibody is added (goat antirabbit IgG) to detect the captured antigen and produce a colored enzyme reaction (Allain et al. Serological markers in early Stages of Human Immunodeficiency virus Infection in Haemophiliacs. Lancet 1986;2: 1233-6). The assay preferentially detects viral p24 antigen (core protein) in early stages of HIV infection, during the

Acquired

immunodeficiency

syndrome

629

“window” period before development iif antienvelope and anticore protein antibodies. Important applications of this test include (1) detection of infectious blood in the 6 to 8 weeks that precede sl:roconversion, (2) determination of congenital HIV infection in children, in whom anti-HIV antibody is i”txsent. which may reflect either infection with HIV or transplacental maternal antibody, and (3? perhaps ii,r prognosis of HIV infection. We thank Dr. Robert F. Carry, ology, for reviewing the manuscript

Department

nf Microbi-

REFERENCES 1. Daul CB, deShazo RD. Acquired immune deticiency syndrome: an update and interpretation. -4nn ~\llergy 198351: 351-61. 2. Norman C. AIDS virology: a battic on mant trrmts Science 1985~2305 18-21. 3. White D, Fenner F. Structure and classification of viruses, In: Medical virology. London: Academic Press Inc, 19863-34. RNA plus genomes that 4. Becker Y, Hadar J. Single-stranded synthesize DNA as part of their life cycle: retroviruses t,RNA tumor viruses). In: Becker Y, Hadar J, eds. Molecular virology. The Hague. Boston. London: Martinus Nyhoff Publishers. 1982:220-49. 5. Becker Y, Hadar J. Molecular considerations or virus replication and virus-cell interactions. In: Becker Y. Radar J, eds. Molecular virology. The Hague, Boston. London: Martinus Nyhoff Publishers, 1982:22-34. 6. Pringle CR. The genetics of viruses In: Topley. Wilson. eds. Principles of bacteriology, virology. and immunity, vol J. 7th ed. Baltimore: Williams & Wilkins, 198359-63. 7. Strayer DR, Gillespie DH. The nature and organization of retroviral genes in animal cells. In: Virology tnooographr, vol 17. New York: Springer-Verlag, 1980. 8. Skehel JJ. Virus replication. In: Topley, Wilson, eds Principles of bacteriology, virology, and immunity. vol. 4. 7th ed. Baltimore: Williams & Wilkins, 1983:49-521. 9. Gallo RC, Wong-Staal F. A human T-lymphotropic retrovirus (HTLV-III) as the cause of the acquired immunodehciency syndrome. Ann Intern Med 1985;103:679-89 10 Wyke JA. Oncogenic viruses. In: Topley, Wilson. eds. Principles of bacteriology, virology, and immunit?. vtll 4. 7th ed. Baltimore: Williams & Wilkins. 19835 1 I -.37 11 White D, Fenner F. Oncogenic viruses. in: White D. Fenner F, eds. Medical virology. London: 4caderntc Press Inc. 1986:217-46. (liTIN-III) lurked 12 Broder S, Gallo RC. A pathogenic tetrovtrus to AIDS. N Engl J Med 1984;311:1292-7 Science 13 Norman C. Patent dispute divides AIDS researchers. 1985:230:640-2. C. A new twist in AIDS patent @ht. Science 14 Norman 1986;232:308-9. 15. Gilden RV, Gonda MA, Sarngadharan MG, Popovic M, Gallo RC. HTLV-III legend correction. Science 19&:232:307. B. et al. First isolation of 16. Gallo RC, Sarin PS, Kramarsky HTLV-III. Nature 1986;321:119. LS, Morrow JW, et al. Infection by the 17. Levy JA, Kaminsky retrovirus associated with the acquired immunodeficiency syndrome. Ann Intern Med 1985;103:694-9. 18. Norman C. What’s in a name? Science 19X5.? ;0:64 1

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19. Coffin .I, Haase A, Levy JA, et al. What to call the AIDS virus? Nature 1986;321:10. 20. Brown F. Human immunodeficiency virus. Science 1986; 232: 1486. 21. Montagnier L. Lymphadenopathy-associated virus: from molecular biology to pathogenicity. Ann Intern Med 1985; 103:689-93. 22. Essex M, Allan J, Kanki P, et al. Antigens of human T-lymphotropic virus type llI/lymphadenopathy-associated virus. Ann Intern Med 1985;103:700-3. 23. Fisher AG, F&berg MB, Josephs SF, et al. The tram-activator gene of HTLV-Ill is essential for virus replication. Nature 1986;320:367-71. 24. Allan JS, Coligan JE, Lee TH, et al. A new HTLV-IIIILAV encoded antigen detected by antibodies from AIDS patients. Science 1985;230:810-13. 25. Sodroski J, Goh WC, Rosen C, et al. A second posttranscriptional tram-activator gene required for HTLV-Ill replication. Nature 1986;321:412-17. 26. Bumy A. AIDS virus: more and better trans-activation. Nature 1986;320:219. 27. Selwyn PA. AIDS: what is now known. Il. Epidemiology. Hosp Pratt 1986;15:127-64. 28. Ho DD, Sarngadharan MG, Resnick L, et al. Primary human T-lymphotropic virus type Ill infection. Ann Intern Med 1985;103:880-3. 29. Gallo RC, Sliski AH. Origin of humanT-lymphotropic viruses. Nature 1986;320:219. 30. Norman C. HTLV-Ill and LAV: similar, or identical? Science 1985;230:643. 31. Marx JL. New relatives of AIDS virus found. Science 1986;232:157.

INSTRUCTIONS

32. Walgate R. AIDS reseatch. New human retroviruses: one causes AIDS .[news] Nature 1986;320:385. 33. Clavel F, Mansinho K, Chamaret S, et al. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N Engl J Med 1987;316:1180-5. 34. Benn S, Rutledge R, Folks T, et al. Genomic heterogeneity of AIDS retroviral isolates from North America and Zaire. Science 1985;230:949-51. 35. Hahn BH, Shaw GM, Taylor ME, et al. Genetic variation in HTLV-lll/LAV over time in patients with AIDS or at risk for AIDS. Science 1986;232:1548-53. 36. Saxinger WC, Levine PH, Dean AG, et al. Evidence for exposure to HTLV-Ill in Uganda before 1973. Science 1985; 227:1036-g. 37. Letvin NL, Daniel MD, Sehgal PK, et al. Induction of AIDSlike disease in macaque monkeys with T-cell tropic retrovims STLV-Ill. Science 230: 1985;230:71-3. 38. Kanki PJ, Alroy J, Essex M. Isolation of T-lymphotropic retrovirus related to HTLV-lll/LAV from wild-caught African green monkeys. Science 1985;230:951-4. 39. Kanki PJ, Barin F, M’Boup S, et al. New human T-lymphotropic retrovims related to simian T-lymphotropic virus type Ill (STLV-lllagm) Science 1986;232:238-43. 40. Palca J. AIDS research. New human retroviruses: and the other does not [news]. Nature 1986;320:385. 41. Harper ME, Kaplan MH, Marselle LM, et al. Concomitant infection with HTLV-I and HTLV-Ill in a patient with T8 lymphoproliferative disease. N Engl J Med 1986;315: 1073-g. 42. Murphey-Corb M, Martin LN, Rangan SRS, et al. lsdlation of an HTLV-Ill-related retrovirus from macaques with simian AIDS and its possible origin in asymptomatic mangabeys. Nature 1986;321:435-7.

FOR EARNING SELF-ASSESSMENT

Pleasenote. The answer sheets for receiving CME credit are no longer included with the self-assessment articles. The self-assessment questions and answers, however, are printed for those wishing to assess themselves. If CME credit is

CATEGORY

1 CREDIT

required, participants can call the American Academy of Allergy and Immunology office (414/272-6071) for answer sheets that should be completed and returned for receipt of Category I credit.

CME examination

Identification

No. 048838

Soto-Aguilar MC, deShazo RD. Human retroviruses and the acquired immunodeficiency syndrome. I. Virology update. J ALLERGYCLIN IMMUNOL 1988;81:619-30.

Question

1

Choose the correct answer:

1. Retroviruses are: a. Single stranded DNA viruses containing a DNA polymerase enzyme b. RNA viruses with a reverse transcriptase enzyme c. DNA viruses with a reverse transcriptase enzyme 630

d. Double stranded DNA viruses containing an RNA polymerase Questions

2-5

Indicate which of the following statements about the life cycle of the retroviruses are true (a) or false

(b): 2. The initial phase of infection by retroviruses oc-