Functional and antigenic characterization of human, rhesus macaque, pigtailed macaque, and murine DC-SIGN.

DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression.

Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.

Coreceptor specificity of temporal variants of simian immunodeficiency virus Mne.

The simian immunodeficiency virus (SIV) Mne envelope undergoes genetic changes that alter tropism, syncytium-inducing capacity, and antigenic properties of the emerging variant virus population during the course of an infection. Here we investigated whether the mutations in envelope of SIVMne also influence coreceptor usage. The data demonstrate that the infecting macrophage-tropic SIVMne clone as well as the envelope variants that are selected during the course of disease progression all recognize both CCR5 and Bob (GPR15) but not Bonzo (STRL33), CXCR4, or CCR3. Although it remains to be determined if there are other coreceptors specific for dualtropic or T-cell-tropic variants of SIVMne that emerge during late stages of infection, these data suggest that such SIV variants that evolve in pathogenic infections do not lose the ability to recognize CCR5 or Bob/GPR15.

Changes in the extracellular envelope glycoprotein of variants that evolve during the course of simian immunodeficiency virus SIVMne infection affect neutralizing antibody recognition, syncytium formation, and macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor recognition.

Simian immunodeficiency virus SIVMne, like human immunodeficiency virus, evolves from a macrophage-tropic, non-syncytium-inducing virus at early times in infection to a T-cell-tropic, syncytium-inducing, cytopathic virus population over the course of progression to AIDS. Because the viruses isolated late in SIVMne infection of macaques include a complex mixture of variants, the viral determinants of such phenotypic changes have not been defined. To identify genetic changes that are important to virus evolution in the host, we constructed chimeric viruses by introducing variant envelope genes representative of proviruses throughout the course of infection and disease into the SIVMne parental clone (SIVMneCL8) that infected the macaque. The chimeric viruses expressed sequences encoding the surface unit of the envelope glycoprotein (Env-SU) of variants cloned between 35 and 170 weeks postinfection. The chimera with Env-SU from 35 weeks postinfection encoded only four changes in V1 compared to SIVMneCL8, whereas the chimeras encoding Env-SU from variants isolated later in infection encoded progressively more mutations both in V1 and elsewhere. Like SIVMneCL8, the chimeras were infectious for CEMx174 cells and macaque peripheral blood mononuclear cells. However, in contrast to SIVMneCL8, the chimeric viruses did not infect macaque macrophages, although each retained the ability to recognize the CCR-5 coreceptor. Thus, these data provide direct evidence that changes which evolve in Env-SU during the course of SIVMne infection do not alter CCR-5 interactions. Viruses encoding Env-SU from the latest times in infection (121 to 170 weeks postinfection), after disease was apparent, were syncytium inducing. However, these viruses were not highly cytopathic, suggesting that additional viral determinants may be required for the rapidly replicating, cytopathic phenotype of the uncloned mixed variant population. Changes in Env-SU did allow the virus to escape serum neutralizing antibodies that recognized the SIVMneCL8 parent. Moreover, the chimera encoding the Env-SU of a virus from 35 weeks postinfection, which differed from SIVMneCL8 only in V1, was not sensitive to neutralization by infected macaque sera, suggesting that V1 may define a portion of the principal neutralizing determinant for SIVMne. Together, these data suggest that SIV variants with changes in the Env-SU may be selected primarily by virtue of their ability to escape neutralizing antibody recognition.

A lymph node-derived cytopathic simian immunodeficiency virus Mne variant replicates in nonstimulated peripheral blood mononuclear cells.

Lymph nodes (LNs) are sites of active human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication and disease at both early and late stages of infection. Consequently, variant viruses that replicate efficiently and subsequently cause immune dysfunction may be harbored in this tissue. To determine whether LN-associated SIVs have an increased capacity to replicate and induce cytopathology, a molecular clone of SIV was isolated directly from DNA extracted from unpassaged LN tissue of a pig-tailed macaque (Macaca nemestrina) infected with SIVMne. The animal had declining CD4+ T-lymphocyte counts at the time of the LN biopsy. In human CD4+ T-cell lines, the LN-derived virus, SIVMne027, replicated with relatively slow kinetics and was minimally cytopathic and non-syncytium inducing compared to other SIVMne clones. However, in phytohemagglutinin-stimulated pig-tailed macaque peripheral blood mononuclear cells (PBMCs), SIVMne027 replicated efficiently and was highly cytopathic for the CD4+ T-cell population. Interestingly, unlike other SIVMne clones, SIVMne027 also replicated to a high level in nonstimulated macaque PBMCs. High-level replication depended on the presence of both the T-cell and monocyte/macrophage populations and could be enhanced by interleukin-2 (IL-2). Finally, the primary determinant governing the ability of SIVMne027 to replicate in nonstimulated and IL-2-stimulated PBMCs mapped to gag-pol-vif. Together, these data demonstrate that LNs may harbor non-syncytium-inducing, cytopathic viruses that replicate efficiently and are highly responsive to the effects of cytokines such as IL-2.

The cytopathicity of a simian immunodeficiency virus Mne variant is determined by mutations in Gag and Env.

Previous studies suggested that the rapidly replicating, highly cytopathic, syncytium-inducing (rapid-high/SI) phenotype of simian immunodeficiency virus Mne variants that evolved in macaques inoculated with a slowly replicating, minimally cytopathic, non-syncytium-inducing (slow-low/NSI) molecular clone was not solely the result of changes in the envelope surface protein (Env SU). To define the viral determinants responsible for the change in phenotype, we molecularly cloned a rapid-high/SI variant (designated SIVMne170) derived from the peripheral blood mononuclear cells (PBMCs) of a pig-tailed macaque that was inoculated with a slow-low/NSI molecular clone, SIVMneCL8. SIVMne170 was SI and replicated with faster kinetics and was more cytopathic than the parent SIVMneCL8 in CEMx174 cells. Additionally, SIVMne170 was more cytopathic for the CD4+ T-cell population than SIVMneCL8 in macaque PBMCs. An analysis of chimeric viruses constructed between the variant SIVMne170 and the parent virus SIVMneCL8 demonstrated that there are determinants encoded within both the 5' and 3' halves of SIVMne170 that independently contribute to its rapid-high/SI phenotype. As we previously observed with other SIVMne variants, the Env SU of SIVMne170 was important for syncytium induction but was not a key determinant of cytopathicity. By contrast, the intracellular domain of the envelope transmembrane protein (Env TM) contributed to both the SI and cytopathic properties of SIVMne170. We also found that the minimal determinant within the 5' half of SIVMne170 that conferred its rapid replication kinetics and cytopathicity mapped to the capsid- and nucleocapsid-encoding regions of gag. Together, these data demonstrate that mutations selected in Gag and Env TM intracytoplasmic tail influence the replication and cytopathicity of SIVMne variants that evolve in the host.

Progression to AIDS in macaques is associated with changes in the replication, tropism, and cytopathic properties of the simian immunodeficiency virus variant population.

Human immunodeficiency virus type 1 (HIV-1) typically evolves from a macrophage-tropic, noncytopathic virus at early asymptomatic stages of infection to a T-cell-tropic, cytopathic, and syncytia-inducing virus population as humans progress to AIDS. This suggests that changes in virus phenotype may influence disease. Because simian immunodeficiency virus (SIV) infection in macaques is a common model system for HIV-1 pathogenesis, we determined whether SIV infection in macaques that develop simian AIDS is associated with a similar shift in viral tropism, replication, and cytopathic properties. The virus that infected the monkeys (SIVMneCL8) and predominated at early times in infection is a macrophage-tropic virus that replicates with relatively low efficiency in human T cell lines. The variant populations that arise in macaques as they progress to AIDS are more infectious for human T cell lines, exhibiting enhanced replication in CEM x 174 cells and an expanded host range that includes Molt-4 Clone 8 cells. Infections starting with equal doses of the viruses demonstrated that the late variants are cytopathic and syncytia-inducing compared to SIVMneCL8, but the variants replicate less efficiently in primary macaque macrophages. V3 sequences were generally conserved between the early and the late variants, suggesting that changes in SIVMne tropism, replication, and cytopathicity were apparently not due to alterations in V3. This study demonstrates important similarities in the phenotypic viral changes that accompany development of AIDS in SIV and HIV-1 infections and suggest that SIV may provide a model system for determining whether the rapidly replicating, T-cell-tropic cytopathic variants present late in infection and disease are indeed important in determining progression to AIDS.

Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I.

The human T-cell leukemia virus type I Tax protein trans-activates several cellular genes implicated in T-cell replication and activation. To investigate its leukemogenic potential, Tax was targeted to the mature T-lymphocyte compartment in transgenic mice by using the human granzyme B promoter. These mice developed large granular lymphocytic leukemia, demonstrating that expression of Tax in the lymphocyte compartment is sufficient for the development of leukemia. Furthermore, these observations suggest that human T-cell leukemia virus infection may be involved in the development of large granular lymphocytic leukemia.

Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones.

Molecular clones of human T-cell leukemia virus type 1 (HTLV-1) have been constructed and stably propagated in plasmids in Escherichia coli. Expression of Tax could be demonstrated from these clones in fibroblast and epithelial cell lines. HOS cells stably transfected with HTLV-1 clone ACH produced all three classes of viral transcripts and Gag proteins. Virus-like particles were also produced from ACH transfected HOS cells as demonstrated by sucrose gradient and electron microscopic analyses. Transfection of peripheral blood mononuclear cells with ACH resulted in production of infectious virus particles which induced lymphocyte proliferation. This study describes useful reagents for further examination of the biological properties of HTLV-1.

CD3-dependent lymphocyte activation by human T cell leukaemia virus type I-producing T cells.

Human T cell leukaemia virus type I-(HTLV-I) transformed cells are capable of stimulating the proliferation of normal T lymphocytes, or stimulating interleukin 2 expression in Jurkat T lymphoid cells. This effect is mediated by the CD2/lymphocyte function-associated antigen 3 (LFA-3) adhesion/signalling pathway. The current work demonstrates that CD3 is also required for this effect, suggesting that the T cell receptor (TCR)/CD3 complex is mediating this effect. However, this effect does not appear to be due to a superantigen since no change in TCR expression was found after HTLV-I-mediated proliferation, nor was proliferation inhibited by an antibody against the specific TCR expressed on Jurkat cells (TCR V beta 8).

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.

Temporal regulation of viral and cellular gene expression during human T-lymphotropic virus type I-mediated lymphocyte immortalization.

An autocrine mechanism of proliferation may play a significant role in the leukemogenesis of adult T-cell leukemia, a mature T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-I). To further delineate the role of HTLV-I and the interleukin 2 (IL2) system in the transformation process, human primary lymphocytes were infected by cocultivation with lethally X-irradiated MT2 cells in the presence or absence of human rIL2; the polymerase chain amplification reaction was used to examine quantitatively the expression of HTLV-I, IL2, and IL2R alpha mRNAs during early and late proliferation phases that displayed polyclonal (days 7 to 49) and oligoclonal (days 100 to 150) proviral integration, respectively. IL2 mRNA and IL2 activity were transiently expressed during the polyclonal phase but were undetectable at later time points. IL2R alpha mRNA expression remained at a constitutively high level throughout the examined time course. Viral transcripts were detectable at each time point. Expression of the tax-rex mRNA was inversely related to IL2 mRNA levels; it was low early in the polyclonal phase but increased 30-fold with the development of oligoclonality. In addition, paraformaldehyde-fixed HTLV-I-producing cells activated peripheral blood lymphocytes. These data suggest that HTLV-I activates human T lymphocytes. However, IL2 expression is transient, indicating a limited involvement of an IL2 autocrine growth loop in the transformation process. Lastly, another viral determinant, in addition to the trans activator tax, may be important in HTLV-I-induced T-cell proliferation.

Replication of human immunodeficiency virus 1 and Moloney murine leukemia virus is inhibited by different heteroatom-containing analogs of myristic acid.

Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational linkage of myristate to the N-terminal glycine residues of several cellular, viral, and oncoproteins. We have recently synthesized a series of sulfur- and oxygen-substituted analogs of myristic acid that are similar in length to the 14:0 fatty acid yet have hydrophobicities equivalent to dodecanoate or decanoate. Previous in vitro enzyme assays and metabolic labeling studies indicate that some of these analogs are excellent substrates for NMT and are incorporated into subsets of cellular N-myristoyl proteins. Their sequence-specific incorporation probably arises from cooperative interactions between the acyl CoA and peptide binding sites of NMT. The human immunodeficiency virus 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) depend on myristoylation of gag polyprotein precursors for assembly. We have tested four analogs--12-methoxydodecanoic acid, 10-propoxydecanoic acid, 5-octyloxypentanoic acid, and 11-ethylthioundecanoic acid--for their ability to block replication of these retroviruses. All reduce HIV-1 replication when incubated with CD4+ H9 cells for 10 days at 10-100 microM. 12-Methoxydodecanoic acid is most effective, producing a concentration-dependent decrease in (i) reverse transcriptase activity (to levels that were 5-10% of control at 20-40 microM), (ii) p24 levels, and (iii) syncytia formation. This degree of inhibition of HIV-1 replication is equivalent to that seen with 5 microM 3'-azido-3'-deoxythymidine and is accomplished without apparent toxicity, as measured by cell viability, protein, and nucleic acid synthesis. 5-Octyloxypentanoic acid inhibits MoMLV assembly in a dose-dependent fashion without accompanying cellular toxicity, while 12-methoxydodecanoic acid has no effect. These data suggest that the use of cellular NMT activity to deliver analogs of myristate with altered physical-chemical properties to proteins that undergo this cotranslational modification may represent an effective anti-viral therapeutic strategy as well as a way to investigate the role of covalently bound fatty acid in viral assembly.

A seroepidemiologic survey of human T-cell lymphotropic virus type I in two Hawaiian hematologic-oncologic practices.

A seroepidemiologic survey for antibodies to the human T-cell lymphotropic virus type I (HTLV-I) was carried out in two Hawaiian hematologic-oncologic practices. Specimens of serum or plasma from 215 donors were assayed using the ELISA technique, followed by the Western blot technique to confirm antibody specificity to HTLV-I. Of 214 seropositive donors, 16 (7.5%) were positive. Of 172 donors of Japanese ancestry, 16 (9.3%) were seropositive; none were white, Chinese, Filipino, or Pacific Islander. One donor contracted the virus through blood transfusions. The results suggest that HTLV-I was introduced to Hawaii with the Japanese immigration.