Regulation of human T cell leukemia virus expression

Retroviruses of the type C morphology have been implicated in a wide variety of diseases in animals and humans. The human T cell leukemia viruses types I (HTLV-I) and II (HTLV-II), the prototypic human-type C retroviruses, have been identified as the causative agents of some forms of human leukemia and neurological disorders. The genetic structure and regulation of the HTLVs are more complex than their avian and murine leukemia virus counterparts. In addition to the gag, pol, and env genes that encode the characteristic virion proteins of all replication competent retroviruses, the genomes of HTLV encode the non-structural proteins, Tax and Rex, which are required for regulating viral gene expression. To understand what appears to be a complex mechanism of disease induction by HTLV, elucidating the regulation and function of the viral gene products and the interaction of these products with each other, as well as with cellular factors, will be critical. This review focuses primarily on regulation of HTLV gene expression in the infected human T lymphocyte, but also discusses analogous gene regulation by the human immunodeficiency virus (HIV). It concentrates specifically on the role these gene products play in virus replication and, ultimately, pathogenesis.     

Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I.

Novel cytoplasmic mRNA species produced by human T-cell leukemia virus type I (HTLV-I) were cloned by using the polymerase chain reaction technique. Five novel 3' splice sites located in the X region and upstream of the env gene were identified. Splicing to the 3' splice sites in the X region generates mRNAs that express two previously unidentified viral proteins, named Rof and Tof. Tof accumulates in the nucleoli of transfected cells. The other viruses of the HTLV family, such as HTLV-II and bovine leukemia virus, also have a complex splicing pattern and are capable of producing additional proteins encoded in the X region. These results suggest that HTLV-I and other members of the HTLV family produce novel proteins, which may contribute to the biological properties of these viruses.     

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 viruses (HTLV-I/II) in South America: Should it be a public health concern?

The presence of the human T cell lymphotropic virus (HTLV-I/II) in South America is well established. Its origin and spectrum in the continent still remain a matter of debate. There are signs now that HTLV-I/II was already present in the Amerindian population coming originally from Asia and that HTLV-I was also introduced with African slave trade and with immigration of individuals from endemic areas of Japan. South America has approximately 350 million inhabitants in its 13 countries. The presence of HTLV-I/II has been reported with impressive numbers in most of them and may be considered endemic in this continent. The distribution of HTLV I/II among native Amerindian populations has shown a geographic clustering of type I in the Andean highlands and Brazilian coast, while type II predominates in lowlands of South America. Although comparability between studies conducted among blood donors in different countries may be difficult, the data indicate that the viruses are also circulating among otherwise healthy individuals. Undoubtedly, HTLV-I/II infection and its related diseases should be considered a public health concern in South America and measures to prevent its spread should be emphasized.     

Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses.

Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses.     

Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements

The Rex regulatory proteins of human T-cell leukemia virus type I (HTLV-I) and bovine leukemia virus (BLV), and the Rev protein of human immunodeficiency virus type 1 (HIV-1), promote the cytoplasmic accumulation and translation of viral messenger mRNAs encoding structural proteins. Rev and Rex act through cis-acting elements on the viral RNA; these elements are named Rev- and Rex-responsive elements, or RRE and RXRE, respectively. We show that the Rex proteins of HTLV-I and BLV are interchangeable, but only the Rex protein of HTLV-I can substitute for Rev of HIV-1. Rex of HTLV-I and Rev of HIV-1 appear to act on RRE by similar mechanisms. Rev of HIV-1 does not act on the RXRE of HTLV-I or BLV. The nonreciprocal action of Rev and Rex suggests that these factors interact directly with the cis-acting RNA elements of the two viruses.     

Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

HIV-1 drug-resistance and drug-dependence

Background

The Deltaretrovirus genus comprises viruses that infect humans (HTLV), various simian species (STLV) and cattle (BLV). HTLV-I is the main causative agent in adult T-cell leukemia in endemic areas and some of the simian T-cell lymphotropic viruses have been implicated in the induction of malignant lymphomas in their hosts. BLV causes enzootic bovine leukosis in infected cattle or sheep. During the past few years several new Deltaretrovirus isolates have been described in various primate species. Two new HTLV-like viruses in humans have recently been identified and provisionally termed HTLV-III and HTLV-IV. In order to identify a broad spectrum of Deltaretroviruses by a single PCR approach we have established a novel consensus PCR based on nucleotide sequence data obtained from 42 complete virus isolates (HTLV-I/-II, STLV-I/-II/-III, BLV). The primer sequences were based on highly interspecies-conserved virus genome regions. We used this PCR to detect Deltaretroviruses in samples from adult patients with a variety of rare T-cell neoplasms in Germany.

Results

The sensitivity of the consensus PCR was at least between 10^-2 and 10^-3 with 100% specificity as demonstrated by serial dilutions of cell lines infected with either HTLV-I, HTLV-II or BLV. Fifty acute T-cell lymphoblastic leukemia (T-ALL) samples and 33 samples from patients with various rare mature T-cell neoplasms (T-PLL, Sézary syndrome and other T-NHL) were subsequently investigated. There were no cases with HTLV-I, HTLV-II or any other Deltaretroviruses.

Conclusion

The results rule out a significant involvement of HTLV-I or HTLV-II in these disease entities and show that other related Deltaretroviruses are not likely to be involved. The newly established Deltaretrovirus PCR may be a useful tool for identifying new Deltaretroviruses.     

Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene.

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.     

A field study of infection with human T-cell leukemia virus among African primates

African non-human primates were surveyed seroepidemiologically for natural infection of human T-cell leukemia virus type I (ATLV/HTLV-I) or its closely related virus(es). Materials from three genera (Cercopithecus, Papio, and Theropithecus), four species (grivet monkey, Anubis baboon, Hamadryas baboon, and gelada), totalling 983 animals under natural conditions, were obtained in a field study in Ethiopia. Virus infection was determined by the indirect immunofluorescence test using HTLV-I specific antigens. Animals seropositive for HTLV-I were found among grivet monkeys and Anubis baboons including the hybrid offspring between Anubis and Hamadryas baboons but not pure-Hamadryas baboons and geladas. From these results, the HTLV-I family was proved to be widespread on the African continent and was regarded as a common retrovirus among catarrhines.     

Mechanism of selection of class II recombinant murine leukemia viruses in the highly leukemic strain CWD

The development of spontaneous lymphomas in CWD mice is associated with the expression of endogenous ecotropic murine leukemia viruses (MuLV) and the formation of recombinant viruses. However, the pattern of substitution of nonecotropic sequences within the envelope genes of the CWD class II recombinant viruses differs from that seen in class I recombinant MuLVs of AKR, C58, and HRS mice. To determine how CWD host genes might influence the envelope gene structure of the recombinant viruses, we characterized the responses of these mice to two different types of exogenous MuLVs. Neonatal mice injected the HRS class I recombinant PTV-1 became infected and developed T-cell lymphomas more rapidly than controls did. The inoculation of CWD mice with the leukemogenic AKR ecotropic virus SL3-3 led to the formation of recombinant MuLVs with a novel genetic structure and class II-like envelope genes, although SL3-3 generates class I recombinants in other strains. These results suggest that the absence of class I recombinant MuLVs in CWD mice is not related to the restriction of the replication or oncogenicity of class I viruses or to the absence of an appropriate ecotropic virus that can generate class I recombinants. More likely, the genes of CWD mice that direct the formation or selection of class II recombinant viruses affect the process of recombination between the ecotropic and nonecotropic envelope gene sequences.     

Construction of a full-length human T cell leukemia virus type I genome from MT-2 cells containing multiple defective proviruses using overlapping polymerase chain reaction

Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.     

Two distinct endogenous type C viruses isolated from the asian rodent Mus cervicolor: conservation of virogene sequences in related rodent species.

The cocultivation of a lung cell line from the Southeast Asian mouse Mus cervicolor with cells from heterologous species has resulted in the isolation of two new distinct type C viruses. Both viruses are endogenous to M. cervicolor and are present in multiple copies in the cellular DNA of these mice. One of the viruses, designated M. cervicolor type CI, replicates readily in the SIRC rabbit cell line and is antigenically related to the infectious primate type C viruses isolated from a woolly monkey (simian sarcoma-associated virus) and gibbon apes (gibbon ape leukemia virus). This virus is also closely related by both immunological and nucleic acid hybridization criteria to a type C virus previously isolated from a second Asian murine species, Mus caroli. The isolation of the M. cervicolor type C I virus thus provides further evidence that the infectious primate type C viruses originated by trans-species infection of primates by an endogenous virus of mice. The second virus, designated M. cervicolor type C II, replicates well in various cell lines derived from the laboratory mouse Mus musculus. While antigenically related to type C viruses derived from M. musculus, the M. cervicolor type C II virus isolate can be readily distinguished from standard murine leukemia viruses. Both new type C viruses from M. cervicolor are unrelated to the previously described retrovirus (M432) isolated from the same Mus species. The DNA of M. cervicolor therefore contains multiple copies of at least three distinct classes of endogenous viral genes. An examination of the cellular DNA of other rodent species for nucleic acid sequences related to the genomes of both M. cervicolor type C I and II reveals that both viruses have been highly conserved evolutionarily, and that other species of rodents, such as laboratory mice and rats, contain endogenous virogenes related to those in the DNA of M. cervicolor.