Human T-lymphotropic virus types I and II.

Human T-lymphotropic virus types I (HTLV-I) and II (HTLV-II) are members of a family of four known retroviruses that are oncogenic as opposed to cytopathic. This family includes HTLV-I and -II, bovine leukemia virus, and simian T-cell leukemia virus. The two types of HTLV are closely related, and for more than a decade we have been aware of the presence of these viruses in humans. In the first part of this article I summarize recent epidemiologic and clinical findings related to the presence of HTLV-I and -II in the Americas. In the second part, I discuss how these viruses may regulate themselves and how in turn they might cause leukemia and neurologic disease in humans.     

Isolation of a novel simian T-cell lymphotropic virus from Pan paniscus that is distantly related to the human T-cell leukemia/lymphotropic virus types I and II.

An unusual serological profile against human T-cell leukemia/lymphotropic virus type I and II (HTLV-I and -II) proteins was reported in several human Pygmy tribes in Zaire and Cameroon with serum antibodies reactive with gp21 and p24. Here we describe a similar pattern of serum antibodies in a colony of captive pygmy chimpanzees and the isolation of a novel retrovirus, simian T-cell lymphotropic virus from Pan paniscus (STLVpan-p), from the peripheral blood mononuclear cells of several seropositive animals. Cocultures of peripheral blood mononuclear cells from three seropositive pygmy chimpanzees with human cord blood mononuclear cells led to the expression of an HTLV-I- and HTLV-II-related virus initially demonstrated by electron microscopy. Furthermore, several of these cocultures became immortalized T-cell lines expressing the CD4+ CD8+ DR+ phenotype of mature activated T cells. Southern blotting and DNA sequencing of a PCR fragment of viral DNA from these cell cultures demonstrated a distant evolutionary relationship of these viruses to HTLV-I and -II and distinct from the known STLV isolates. We designated this virus STLVpan-p. A genealogical analysis of the captive pygmy chimpanzees colony, originated from wild-caught animals, revealed a prevalence of seropositive offspring from infected mothers, as also observed with HTLVs. The presence in this old African Great Ape species of a virus which is genetically quite distinct from HTLV-I and -II could provide new insights in the phylogenesis of STLVs and HTLVs and be instrumental in the discovery of related human viruses.     

Human T-cell lymphotropic virus type I and adult T-cell leukemia/lymphoma outside Japan and the Caribbean Basin

Ninety-six patients with the diagnosis of adult T-cell leukemia/lymphoma (ATLL) were identified in countries outside Japan and the Caribbean Basin. Seventy-four of these patients were initially diagnosed in the United States; 25 of 52 patients whose places of birth were known had been born in the United States. The detection of 14 patients born in the southeastern United States, all black, indicates a group deserving particular attention for studies of human T-cell lymphotropic virus type I (HTLV-I), a suspected etiologic agent in most cases of ATLL. Although geographic clustering of ATLL in areas endemic for HTLV-I, particularly southwest Japan and the Caribbean Basin, is a dramatic feature of this disease, a review of the literature indicates that HTLV-I-associated ATLL probably occurs sporadically in a much wider distribution, the disease being diagnosed in native-born African, Chinese, European, and Latin American patients. A registry for ATLL cases is suggested, to assist in the identification of risk factors for this disease and, at the same time, improve case definitions and early diagnosis.     

Human T-cell leukemia virus type I envelope protein maturation process: requirements for syncytium formation.

The human T-cell leukemia virus type I (HTLV-I) envelope protein is synthesized as a gp61 precursor product cleaved into two mature proteins, a gp45 exterior protein and a gp20 anchoring the envelope at the cell membrane. Using N-glycosylation inhibitors and site-directed mutagenesis of the potential glycosylation sites, we have studied the HTLV-I envelope intracellular maturation requirements for syncytium formation. We show here that experimental conditions resulting in the absence of precursor cleavage (tunicamycin, monensin treatments, and use of inhibitors of the reticulum steps of the N glycosylations) also result in no cell surface expression of envelope protein. The lack of syncytium formation observed in these cases is thus explained by incorrect intracellular transport. When the precursor is cleaved in the Golgi stack (no treatment or treatment with inhibitors of the Golgi steps of the N glycosylations), it is transported to the cell surface in all the cases examined. Syncytium formation is markedly reduced, however, when Golgi glycosylations are incorrect, which shows that the sugar moieties are involved in the envelope functions. Site-directed mutagenesis demonstrates that each of the five potential glycosylation sites is actually glycosylated. Glycosylation of sites 1 and 5 is required for normal maturation, whereas that of sites 2, 3, and 4 is dispensable. Glycosylation of each site, however, is required for normal syncytium formation. Altogether, the restraints exerted by the cell for the HTLV-I envelope to be transported and functional are very high, which might play a role in the observed conservation of the envelope amino acid sequence between various strains.     

Multiple isolates and characteristics of human T-cell leukemia virus type II.

Human T-cell leukemia (or lymphotropic) virus type II (HTLV-II) was isolated from eight HTLV-seropositive patients, six of whom were also infected with human immunodeficiency virus, by cocultivation of peripheral blood mononuclear cells (PBMCs) with BJAB, a continuous B-cell line. Restriction endonuclease mapping of the proviruses demonstrated consistent differences among isolates, and two distinct physical map patterns were observed. The results suggest the existence of two closely related molecular subtypes of HTLV-II, which are tentatively designated HTLV-IIa and HTLV-IIb. This finding was supported by preliminary nucleotide sequence analysis of the env gene region encoding the transmembrane glycoprotein gp21, which showed consistent differences between the two proposed virus subtypes. Exploitation of differences in restriction endonuclease sites allowed polymerase chain reaction amplification to detect and differentiate the two subtypes in fresh PBMCs of HTLV-seropositive intravenous drug abusers (IVDAs). The results of these studies confirm that HTLV-II infection is the prominent HTLV infection in seropositive IVDAs and also show that infection with both subtypes occurs. The finding of genetic heterogeneity in the HTLV-II group of viruses may have important implications for studies on its role in human disease and will be useful in characterizing the viruses present in newly discovered endemic foci in New World indigenous populations.     

Intrauterine transmission of human T-cell leukemia virus type I in rats.

To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.     

Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II.

DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.     

Transcomplementation of simian immunodeficiency virus Rev with human T-cell leukemia virus type I Rex.

A molecular clone of the simian immunodeficiency virus SIVSMM isolate PBj14, lacking the ATG initiation codon for Rev protein (PBj-1.5), did not produce virus or large unspliced or singly spliced viral RNA upon transfection of HeLa cells. Low but significant levels of virus and large viral RNA production were observed upon transfection of PBj-1.5 into HeLa Rev cells expressing the rev gene of human immunodeficiency virus type 1. Furthermore, abundant virus and large viral RNA production occurred upon transfection of PBj-1.5 into HeLa Rex cells expressing the rex gene of human T-cell leukemia virus type I. Virus produced from HeLa Rex and HeLa Rev transfections was infectious, produced large amounts of virus, and was cytopathic for Rex-producing MT-4 cells. In contrast, no or only low levels of virus production were observed upon infection of H9 cells. These studies show that a defective SIV rev gene can be transcomplemented with human immunodeficiency virus type 1 Rev and with high efficiency by human T-cell leukemia virus type I Rex, and they suggest that rev-defective viruses could serve as a source for production of a live attenuated SIV vaccine.     

The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments.

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.