Molecular Biology and Pathogenesis of Animal Lentivirus Infections

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, Jan. 1996, p Vol. 9, No /96/$ Copyright 1996, American Society for Microbiology Molecular Biology and Pathogenesis of Animal Lentivirus Infections JANICE E. CLEMENTS* AND M. CHRISTINE ZINK Division of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland INTRODUCTION HISTORICAL PERSPECTIVE BIOLOGICAL SIMILARITIES MOLECULAR BIOLOGY VIRUS REPLICATION REGULATION OF LENTIVIRUS GENE EXPRESSION BY VIRAL PROTEINS ROLE OF CELL TROPISM IN THE PATHOGENESIS OF UNGULATE LENTIVIRUSES ROLE OF CELL TROPISM IN SIV PATHOGENESIS MACROPHAGE-TROPIC STRAINS OF SIV ELICIT CROSS-PROTECTIVE IMMUNE RESPONSES CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES INTRODUCTION With the emergence of human immunodeficiency virus (HIV) as the causative agent of AIDS in humans, lentiviruses have moved from the position of a rather obscure group of animal viruses to become one of the most studied groups of viruses. All of the lentiviruses have a number of clinical features in common: long incubation periods, persistence in the face of vigorous immune responses, multiorgan disease, and an invariably fatal outcome. The lentiviruses can be divided into two groups on the basis of their tropism for different host cells. Equine infectious anemia virus (EIAV), the ovine lentiviruses (OvLV; including ovine progressive pneumonia viruses and maedi-visna virus), and the caprine lentiviruses (strains of caprine arthritis-encephalitis virus [CAEV]) replicate predominately in macrophages. In contrast, the human, simian, and feline viruses replicate in both lymphocytes and macrophages. This difference in cell tropism is responsible for the differing disease manifestations of these two virus groups. Though the clinical manifestations of infection by individual lentiviruses are often quite different, the underlying mechanisms of persistence and pathogenesis are surprisingly similar. The unique pathogenesis of the lentiviruses is attributable to both their complex genetic structure and the novel molecular mechanisms controlling viral gene expression. Lentiviruses have similar genetic compositions, with the structural genes gag, pol, and env encoding the core proteins, reverse transcriptase, and envelope glycoproteins, respectively. In addition, regulatory genes modulate the viral life cycle in vitro and probably contribute to clinical latency and pathogenic mechanisms in vivo. The regulatory genes tat and rev control viral transcription and RNA transport and translation, vif and vpu regulate production of infectious viral particles, and vpr and nef are involved in disease manifestations. These regulatory genes share little nucleotide or amino acid homology from one lentivirus to another, yet their functions are conserved. These genes may have been transduced from the host genome, supplying functions essential to the lentivirus for persistence and * Corresponding author. Mailing address: Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Ave., Traylor G-60, Baltimore, MD Phone: (410) Fax: (410) pathogenesis in vivo. In this review, the molecular biology, cellular tropism, and pathogenesis of the lentiviruses will be discussed, using two animal model systems as examples. OvLV will be discussed as a prototype for lentiviruses that are mainly macrophage-tropic, and simian lentivirus infection of rhesus macaques will be used as an example of a lentivirus that replicates in both macrophages and lymphocytes. HISTORICAL PERSPECTIVE EIAV was not only the first lentivirus to be identified but also the first nonplant virus to be discovered (203). This happened at the turn of the century, close on the heels of the discovery of the first virus, tobacco mosaic virus. EIAV causes chronic, relapsing anemia in horses, a condition that had been recognized in Europe since 1843 (116). It was an unfortunate twist of fate that for the next half-century EIAV proved to be impossible to culture in vitro. As a result, research on the pathogenesis, immunology, and molecular biology of this virus lagged behind that of other lentiviruses. Lentiviral disease in sheep was first described as a progressive pneumonia of sheep in South Africa by Mitchell in 1915 (159). This was followed in 1923 by a report of severe chronic interstitial pneumonia of sheep in Montana that resulted in wasting and eventual death (129). About 15 years later, a new form of chronic pneumonia (termed maedi) was recognized in Icelandic sheep by Gislason (71). During the next decade in Iceland, a neurological condition characterized by paralysis and wasting (termed visna) was identified only in flocks that also had maedi (159). Through epidemiological studies, it became evident that the emergence of maedi-visna in Iceland had occurred subsequent to the introduction of 20 Karakul rams from Germany. Maedi-visna became the subject of intensive epidemiological, pathological, and virological study in Iceland. These studies led Bjorn Sigurdsson to conclude that the condition was caused by a slow virus that, unlike conventional viruses, manifested its effects over months and years (188). This term led to the classification of these viruses as lentiviruses (lenti, slow). Since that time, lentiviral diseases of sheep have been recognized worldwide and are called by a variety of names including maedi-visna virus, ovine progressive pneumonia virus, and OvLV. Infection of goats with lentivirus was first recognized in

2 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 101 as the result of an epizootic of leukoencephalomyelitis in kid goats 1 to 4 months of age in the northwestern United States (36). Epidemiological and virological studies of goat herds with a high incidence of arthritis and leukoencephalomyelitis revealed that the same virus caused a chronic proliferative synovitis and arthritis in adult goats and leukoencephalomyelitis in kid goats, and the agent was named CAEV (1, 30, 36, 38, 150). The epizootic of CAEV that occurred in these herds was not caused by the introduction of a new virus but was initiated by a recent change in herd management that resulted in increased spread of preexisting, unrecognized CAEV. Dissemination of CAEV, normally transmitted from dam to kid in colostrum and milk, was amplified by feeding newborn goats milk pooled from several animals. Since that outbreak, serological studies have shown that CAEV is widely disseminated throughout North America, Europe, Australia, and New Zealand (1). During the late 1960s, a number of laboratories were searching for the etiological agent of bovine leukemia-lymphosarcoma in hopes that this would lead to a further understanding of virus-induced carcinogenesis. A number of bovine retroviruses were identified as a result of this research effort, including one that morphologically resembled maedi-visna virus of sheep (204). More than 15 years passed before this virus was studied in detail and given the name bovine immunodeficiency virus (73, 75). This study linked bovine immunodeficiency virus with other immunodeficiency-causing lentiviruses that had been discovered in the meantime, of which HIV type 1 (HIV-1) and HIV-2 were the most notable members. HIV-1 was first identified in 1983 as the agent responsible for an immunodeficiency syndrome that led to opportunistic infections and death in gay men (10, 57). It soon became evident that this newly discovered virus was morphologically and genetically related to the other known lentiviruses, maedivisna virus and EIAV (74, 76), and that, as with the other lentiviruses, HIV-1 caused multiorgan disease inexorably leading to death. Several years after the discovery of HIV-1, another HIV, HIV-2, was discovered in Africa (27). During the decade after the discovery of HIV-1, a number of lentiviruses were identified by seroepidemiology and virus isolations from several species of nonhuman primates. Collectively, these lentiviruses have been termed the simian immunodeficiency viruses (SIVs); this group is composed of lentiviruses that infect different species of nonhuman primates (the subscript denotes the species from which the virus was isolated) including macaques (SIV mac ) (12, 43), the sooty mangabey (SIV sm ) (54, 146), the African green monkey (SIV agm ) (99, 156), the mandrill (SIV mnd ) (202), the Sykes monkey (SIV syk ) (87), and the chimpanzee (SIV cpz ) (92, 164). Many of the lentiviruses from nonhuman primates were isolated from animals caught in the wild, and these animals showed no signs of any clinical disease. In contrast, SIV mac was isolated at the New England Primate Center from a rhesus macaque with lymphoma (93). The virus was shown to cause an AIDS-like syndrome when inoculated into other rhesus macaques (43). Studies of wild rhesus monkeys of Asian origin have suggested that these monkeys do not naturally harbor the virus (120, 156). There have been additional isolations of SIVs from different macaque species associated with lymphoma at other primate centers (Davis, Calif.; Yerkes, Atlanta, Ga.; Tulane, Covington, La.; and Seattle, Wash.), (12, 54, 104, 146), and comparisons of these viruses suggest that the SIV isolates that cause disease arose from cross-species transmission from mangabeys into macaques (44, 88). In their natural hosts the simian lentiviruses appear to be relatively nonpathogenic; however, the transfer of these viruses into other species naturally free of the virus results in disease. This has been observed previously with the epidemic of visna-maedi disease in Iceland after the introduction of foreign sheep (see above) and may also account for the emergence of AIDS in Africa. This is supported by the genetic relationship of the human virus HIV-2 to SIV mac and SIV sm isolates and of HIV-1 to the SIV cpz isolates (88). Feline immunodeficiency virus (FIV) is the most recently identified immunodeficiency-causing lentivirus. FIV is frequently found in feral cats that are infected with both FIV and another (oncogenic) retrovirus that induces immunodeficiency, feline leukemia virus (163). FIV was first isolated in 1986 from a feline leukemia virus-negative cattery in California with a high incidence of cats that suffered from an immunodeficiencylike syndrome (163), and it is now recognized as a major cause of immunodeficiency in cats in North America. Lentiviruses can be relatively nonpathogenic in their natural hosts, as evidenced by widespread infections of nonhuman African primates with strains of SIV and worldwide infections of goats and sheep with lentiviruses. Lentiviral infections do not always result in clinically evident diseases, and diseases, when manifested, have long incubation periods. Thus, it is conceivable that there are other species that harbor specific lentiviruses, and these may be the causative agents for other chronic-progressive disease syndromes. BIOLOGICAL SIMILARITIES The lentiviruses have biological properties distinct from those of other retroviruses (149). They replicate in nondividing, terminally differentiated cells and are relatively species specific, usually replicating only in cells that originate from their natural host or from closely related species. This species specificity has hampered the development of animal models for HIV-1-induced disease and has necessitated the use of other lentiviruses as models for HIV pathogenesis, vaccine production, and drug development. HIV-1 has been shown to infect some nonhuman primates such as chimpanzees (54) and pigtail macaques (2); however, there is no evidence of disease in these animals. SCID (severe combined immunodeficient) mice have been reconstituted with human lymphoid tissue or human peripheral blood mononuclear cells, but again, there is no disease in these animals (132, 144). One exception to the relative species specificity of the lentiviruses is bovine immunodeficiency virus. Recent studies have established both a rabbit model and a transgenic mouse model for bovine immunodeficiency virus-induced disease (75, 168). The major host cells of the lentiviruses are cells of monocyte/ macrophage lineage and CD4 lymphocytes. Infection of these immune cells is the basis for the multiorgan disease that is characteristic of all lentiviral infections (25, 35, 64 66, 141, 149, 184). Although there is a long delay between initial infection and the development of disease, considerable virus replication occurs in the first few weeks after infection, and during this acute phase the lentivirus spreads throughout the host. Primary sites of viral replication include the lymph nodes, spleen, and bone marrow. Virus replication in these tissues serves as a reservoir for the distribution of infected monocytes and lymphocytes to multiple sites in the body, including the brain, lung, joints, and other organs where the monocytes mature into macrophages and the lymphocytes are activated (64 66, 184, 221). Differentiation-activation of the cells activates viral gene expression, and virus is then produced in these tissues (55, 65, 66, 170, 183). This ability to remain latent is one mechanism that the virus uses to evade detection by the host and is one reason why the manifestations of clinical disease and produc-

3 102 CLEMENTS AND ZINK CLIN. MICROBIOL. REV. tion of significant virus-induced pathology take months to years to develop (48, 49, 149). Another strategy that lentiviruses use to evade the host immune responses is genetic variation. Animals inoculated with cloned virus strains eventually develop a highly divergent population (viral swarms) of viral genomes; this has been found in infections with the animal lentiviruses visna virus, CAEV, EIAV, and SIV (19, 24, 96, 106, 134, 151, 177). Antigenic variation has also been observed in humans infected with HIV-1 (13, 81, 137, 172). The development of antigenic variants can be directly linked to episodes of fever and viremia in horses infected with EIAV (28, 106, 178). However, the role of antigenic variant viruses in the progression of other lentivirus diseases is unclear. Antigenic variation of the envelope glycoprotein of visna virus may provide a mechanism for the virus to persist and continuously replicate in an immunocompetent host (28). Antigenically distinct variants of EIAV, SIV, and visna virus are fully virulent and cause disease when inoculated into naive animals. Thus, the selection of variants in vivo appears to directly contribute to disease in EIAV-infected animals and may contribute to the continued virus replication and eventual disease manifestation in SIV- and visna virus-infected hosts. MOLECULAR BIOLOGY Retroviruses contain three genes, gag, pol, and env, that encode the viral structural and enzymatic proteins. In contrast, the lentiviruses have a more complex genetic organization containing additional small open reading frames (ORFs) located between the pol and env genes, as well as exons contained within and at the 3 end of the env gene (39, 40). In addition, HIV-1 has the largest number of these ORFs characterized (Fig. 1). Six ORFs (vif, vpu, vpr, tat, rev, and nef) have been identified in the HIV-1 genome (40, 182). HIV-2 and SIV mac have a vpx gene not found in HIV-1, but they lack the vpu ORF (44, 52). The ungulate lentivirus genomes appear to have fewer of the small ORFs found in the primate viruses. Visna virus (as the prototype of OvLV) and CAEV are closely related biologically, and their genetic organizations are very similar: they contain ORFs that correspond to vif, tat, and rev (15, 179, 193). The location of these ORFs also differs from that of the ORFs in the primate lentiviruses (Fig. 1). vif, tat, and rev ORFs have also been identified in the genome of EIAV (101, 176, 196). The additional gene products of the lentiviruses contribute to a more complex pattern of gene expression and may also contribute to the pathogenesis of disease. In cell culture, lentivirus replication can be separated into early and late gene expression (39). Early gene expression is characterized by the presence of the Tat, Rev, and Nef proteins, all products of multiply spliced viral mrnas. In contrast, the structural, enzymatic, and accessory proteins Vif, Vpu, and Vpx are synthesized from unspliced or singly spliced viral mrnas during late gene expression. A brief overview of the function of the accessory proteins of the lentiviruses is presented here. The role of the Tat and Rev proteins is considered below in the section on the control of virus gene expression. The nef gene is present only in the primate lentiviruses and is not required for virus replication in cell culture (103). However, both the SIV and HIV-1 nef genes appear to be required in vivo for efficient virus replication and disease progression (42, 95, 103). The nef gene of SIV mac s that have point mutations is found to revert rapidly in vivo to a wild-type nef gene, and SIV mac s with deletions in the nef gene cause infection but not disease in rhesus macaques (42, 103). Rhesus macaques infected with SIV mac with a nef deletion are protected against infection with wild-type virulent virus (42). The mechanism of action of the Nef protein is not completely understood; however, the expression in cells of either the HIV-1 or SIV Nef proteins results in the downregulation of surface CD4 (the cellular receptor for both viruses) (4, 11, 58, 59, 180). Thus, the nef gene appears to function by reducing the amount of CD4 on the surface of infected cells, thereby facilitating the expression of the Env proteins on the cell surface without interactions with CD4. The Nef protein is clearly important in the pathogenesis of SIV and may also be important in HIV-1. HIV-1 Nef expression in transgenic mice resulted in developmental abnormalities in CD4 lymphocytes (117, 190). It is curious that the strictly monocyte/macrophagetropic lentiviruses do not contain a unique ORF for the nef gene. This lack may be related to the use of receptors other than CD4 which may have lower affinities than CD4 and, thus, not require the downregulation of the receptor on the surface of the infected cell. Alternatively, these other lentiviruses may have gene products that function in a fashion analogous to the Nef protein, and once the receptors for these viruses are identified, this function may be found intrinsic to one of the virus proteins. In bovine immunodeficiency virus, a truncated form of the transmembrane protein is expressed, and Garvey et al. have postulated that this protein may be the Nef equivalent in the nonprimate lentiviruses (61). The Vif (viral infectivity factor) protein, although not universally found in the lentivirus virion, appeared in early studies to facilitate the infectivity and spread of virus, particularly in primary lymphocyte and macrophage cultures (98, 112, 155, 192). More recent studies showed that the effects of the Vif protein were on early events (proviral DNA synthesis) after viral infection (194, 205). This finding seems contradictory if the Vif protein is not in the virion. However, studies show that vif affects the proper assembly of the nucleoprotein core (56, 72, 140), which in turn may affect whether virions are functional after infection. Thus, the complete mechanism of Vif action remains to be understood. The vpu gene is found in the virion of HIV-1 but not in other lentivirus genomes (197). The function of this protein in the virus life cycle is currently not clear. However, mutations in the vpu gene result in virus particles with multiple cores that bud into cell vacuoles rather than from the cell surface (105, 211, 212). In addition, it has been suggested that the Vpu protein forms an ion channel and is important in the formation and release of viral particles (105, 211). In this regard, the Vpu protein has been shown to be a type 1 integral membrane protein and forms oligomers in the infected cell (125). Another function of the Vpu protein is the degradation of CD4, the cellular receptor for HIV-1 and HIV-2 (70, 113, 207). The vpr gene is conserved in the genomes of HIV-1, HIV-2, and most strains of SIV. The protein is present in the virion, suggesting that it may play a role early after infection of the cell (33, 34, 83, 114, 214, 215). For HIV-2, the vpr gene has been shown to be required for viral replication in primary monocyte/ macrophage cells, suggesting an important role for this protein in vivo (83, 114). Further, a recent study on the vpr gene of HIV-1 has demonstrated that it can induce differentiation in vitro in rhabdomyosarcoma cell lines derived from muscle tumors (114). The Vpr protein localizes in the nucleus of cells (121); its role early after infection appears to be in the efficient localization of the preintegration complex in the nucleus of macrophages (85). Thus, the Vpr protein provides a function similar to the nuclear localization signal of the p17 matrix protein (present in the virion core) and provides a duplicate

4 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 103 FIG. 1. Genetic organization of selected lentiviruses.

5 104 CLEMENTS AND ZINK CLIN. MICROBIOL. REV. mechanism for the primate lentiviruses to integrate proviral DNA into the genome of nondividing cells (17, 80). The vpx gene is unique to HIV-2 and the SIV group of lentiviruses (Fig. 1). It is interesting that its product shares considerable homology with the Vpr protein of HIV-1 and is found in the virus particle (201, 213). Vpx appears to affect early proviral synthesis in infected primary cells (34, 100). The precise mechanism of action of Vpx is unclear, but it appears to affect the efficiency of early replicative events. VIRUS REPLICATION Lentiviruses are enveloped RNA viruses that bud from the cellular membrane, producing mature particles approximately 100 nm in diameter. The lentivirus virion core structure is cylindrical; this structural feature distinguishes this group of viruses from the other retroviruses (74, 76). The virion core is composed of the gag proteins p24, p17, p9, and p7 (these sizes are slightly different for each lentivirus) that are proteolytically processed from a 53- to 55-kDa gag precursor protein by the virion-encoded protease. The virion core contains two copies of viral RNA associated with the p7 protein. This protein contains zinc finger motifs in all lentiviruses examined and probably interacts with the viral RNA via these protein motifs (77, 79). The viral reverse transcriptase and integrase are also associated with the viral RNA. Recently, a new gene segment has been located in the pol gene between RNase H and the integrase in nonprimate lentiviruses (133). The genomes of FIV, EIAV, visna virus, and CAEV contain this genetic element, but it is lacking in the pol genes of the primate lentiviruses. This region encodes a functional dutpase activity in FIV (47), EIAV (199), and visna virus (162) which has been found in gradient-purified virions, indicating that these viruses bring this protein into the cell upon infection. Analysis of this gene segment in EIAV has provided evidence that deletion of the dutpase domain in an infectious clone results in production of functional reverse transcriptase and a virus that replicates to wild-type levels in continuous cell lines. However, the dutpase-deficient EIAV replicated poorly in cultures of primary equine macrophages. These data suggest that the virus-encoded dutpase is required for efficient replication in macrophages, which are nondividing, terminally differentiated cells. The function of the dutpase gene segment in the genomes of nonprimate lentiviruses is unclear at this time. However, because the monocyte/macrophage is the primary target cell for these viruses in vivo, this virus-encoded enzyme activity may reflect the adaptation to replication in these nondividing cells. Cellular dutpases are cell cycle regulated; one of their roles in DNA replication in cells is thought to prevent uracil incorporation into DNA (46, 136). In this regard, dutpasedeficient EIAV efficiently incorporates uracil into reverse-transcribed products in vitro, while wild-type EIAV does not (162). Thus, it is attractive to speculate that the EIAV dutpase may prevent uracil incorporation into DNA during replication of virus in nondividing cells, where cellular dutpase levels might be low. The virion core is surrounded by the viral envelope that is derived from the cellular membrane in which the viral surface and transmembrane proteins have been inserted. There is a considerable body of information on the synthesis, maturation, and epitope organization of the envelope proteins of HIV-1; however, this topic is beyond the scope of this review, and the interested reader is directed to recent reviews (51, 131, 139, 142). Briefly, the envelope proteins of the lentiviruses are synthesized as a large precursor protein that is cleaved by a cellular protease into the surface (gp120 to gp135) and transmembrane (gp40 to gp50) glycoproteins. The transmembrane glycoprotein has two hydrophobic domains; one at the amino terminus is responsible for virus-induced cell fusion and is only functional after cleavage of the glycoprotein precursor. The second hydrophobic domain spans the cellular membrane and anchors the glycoproteins in the membrane. The surface glycoprotein is highly glycosylated and forms the spikes observed on the virion surface; it contains the epitopes that interact with the cellular receptor as well as the determinants for virus neutralization. The first step in the viral life cycle is the interaction of the virion with the target cell; the surface glycoprotein of the virus interacts with specific receptors on the cell surface. The major cellular receptor for the primate lentiviruses is the CD4 molecule on the surface of T lymphocytes and monocytes/macrophages (123). Recently, another molecule, galactosyl ceramide, has been identified as a potential receptor for HIV-1 in cells derived from the central nervous system (14, 82). Identification of receptors for other lentiviruses is still tentative. Two recent reports have identified two putative receptors for visna virus, one suggesting that sheep class II histocompatibility antigen may be involved in virus-cell interactions and the other identifying a cell surface molecule of 50,000 kda (37, 41). Interaction of lentiviruses with their receptors apparently causes a conformational change in the transmembrane envelope glycoproteins that exposes the hydrophobic domain at the amino terminus of the protein. Viral entry appears to be mediated through the fusion of the viral envelope with the cellular membrane (124, 195). After virus entry into the cell, the virus is partially uncoated and the viral RNA is copied by the virion-associated reverse transcriptase, generating a double-stranded DNA copy of the viral RNA genome. In the early steps of HIV-1 DNA replication in T lymphocytes, activation of the cell is apparently required for complete synthesis of the double-stranded DNA (216). In addition, cellular factors play a crucial role at these early replication steps since there is a host restriction observed for many lentiviruses at this stage (123). Despite the introduction of the human CD4 gene into mouse cells, HIV-1 is unable to infect such cells; this host restriction can be overcome by transfecting the viral DNA into such cells. This results in a single replicative cycle with no reinfection of the cell by the progeny virus (50, 115). These results suggest that host-specific factors may be required for postbinding virus entry or in proviral DNA synthesis. The termini of the long terminal repeats (LTRs) that flank the viral DNA genome are involved in the integration of the viral DNA into the host chromosome mediated by the viral integrase (Fig. 2). Once the viral DNA is integrated into the cellular genome, the cell may either remain latently infected, with little or no expression of virus, or be productively infected. This step could be controlled at the level of cellular activation. Recent evidence suggests that a critical functional threshold of the regulatory gene rev is required to shift a latently infected monocytic cell line (U1) to a productive infection (see below). The LTRs also contain signals for transcriptional activation as well as RNA synthesis, capping, and polyadenylation (Fig. 2). Sequences that interact with cellular transcription factors are found in the U3 region of the LTRs. Transcription of lentiviral genomes is initially dependent on the presence in the cell of the appropriate cellular transcription factors. The specific cellular transcription factors utilized by a particular lentivirus are in part determined by its cellular tropism. The immunodeficiency viruses contain the binding sites for the transcription factors NF- B and SP1 (53, 60, 97, 148), whereas

6 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 105 FIG. 2. Transcriptional control elements of lentiviruses. The LTR contains the U3, R, and U5 regions. The U3 region contains the transcriptional regulatory sequences called enhancer and promoter elements. The R region contains the cap site, the start of mrna synthesis, and the signal for polyadenylation. The core enhancer element is shown at the bottom for two groups of lentiviruses, the macrophage-tropic (visna virus and CAEV) and the lymphocyte-macrophagetropic (HIV-1, HIV-2, and SIV) viruses. the macrophage-tropic viruses (visna virus and CAEV) contain AP-1 and AP-4 binding sites (55, 63, 86, 179). The binding of NF- B to the HIV-1 LTR and cellular factors to the AP-1 binding sites in the visna virus LTR are important for transcriptional activation of these viruses in T-cell and macrophage cell lines, respectively. In addition to these sites, both viruses have sites in the U3 regions that are recognized by other cellular transcription factors (55, 210, 217). Thus, one of the cellular factors that activates transcription of HIV-1, HIV-2, and SIV is NF- B, a transcription factor present in activated T lymphocytes and monocytes (62). In contrast, visna virus, CAEV, and FIV utilize the transcription factors c-jun and c-fos (that bind to AP-1 sequences) found in activated or differentiated macrophages (55, 186). Recently, the monocyte/ macrophage-specific transcription factor Pu.1 has been shown to be a transcriptional regulator of the EIAV LTR (23). In addition to these well-characterized transcription factors, there are sites for additional cellular factors in the U3 region of all lentiviruses. These factors may provide lentiviruses with the capacity to be activated at the transcriptional level in many different cellular environments. The U3 region of the lentivirus genome has a high degree of heterogeneity, which may allow for selective replication of particular viruses in specific cells and organs. Upstream in the U3 regions of HIV-1, visna virus, and EIAV, a negative regulatory element has been described (86, 121, 187). A cellular factor that binds to the HIV-1 negative regulatory element has been found in murine and human cells, and the gene has recently been cloned (20). In addition to sequences that control RNA transcription, the R regions of HIV-1, HIV-2, SIV, and EIAV contain direct repeats that form stable stem-loop structures in the nascent RNA molecules (8, 9, 22, 67, 84, 145, 174, 185). This structural element appears to be critical for the action of the regulatory protein Tat of these viruses and has been called the TAR (Tat activating region) element. However, in visna virus, CAEV, and FIV, no TAR structure can be identified by comparative computer analysis, and the tat genes of visna virus and CAEV appear to act independently of such a structure (15, 86, 179, 198). The ability of lentiviruses to replicate in nondividing cells distinguishes them from other groups of retroviruses. The presence of a group of auxiliary genes that are dispensable for replication of lentiviruses in some cells in vitro but not in others, as well as the highly conserved nature of these ORFs in HIV-1 and the other lentiviruses, suggests that these gene products may play critical roles in lentivirus replication in vivo. To date, the nef and vpr genes in SIV have been shown to be dispensable for replication in cell lines in vitro but contribute to virus titer and pathogenicity in vivo (103, 111). The vpr genes of the primate lentiviruses have been implicated in nuclear localization of the preintegration complex. This gene, along with nucleophilic determinants in the gag gene p17 (matrix protein), provides mechanisms for lentiviruses to integrate the proviral DNA in a nondividing cell (17, 18, 80, 85). It is possible that the presence of different subsets of auxiliary genes confers species specificity and unique pathobiology to each member of the lentivirus group. REGULATION OF LENTIVIRUS GENE EXPRESSION BY VIRAL PROTEINS Lentivirus gene expression is characterized by early and late phases; however, transcription of the viral RNA always results in the synthesis of a full-length mrna species that serves as the viral genome as well as the mrna for the gag and pol genes. Processing of the full-length viral mrna occurs in the nucleus of the infected cell, and the levels of spliced and unspliced viral mrnas that are in the cytoplasm of the cell are controlled by the viral regulatory proteins Tat and Rev. During the early phase of viral gene expression, the only viral transcripts present in the cytoplasm are multiply spliced viral mrnas that encode the regulatory proteins Tat, Rev, and Nef. In contrast, during the late phase, unspliced and singly and multiply spliced viral RNAs are present in the cytoplasm. The tat gene of lentiviruses functions to increase viral gene expression at both the transcriptional and posttranscriptional levels (31, 40). In some lentiviral RNAs (HIV-1, HIV-2, SIV, and EIAV), the stem-loop structure called TAR, located at the 3 end of the viral mrna, serves as a binding site for the Tat protein and for cellular proteins that function to increase gene expression from the viral promoter and to increase the processivity of RNA transcription by the cellular RNA polymerase II. In contrast, lentiviruses that lack a TAR element (visna virus, CAEV, and FIV) have Tat proteins that act in a more indirect fashion (63). Recent data obtained for the visna virus Tat protein suggest that it is a transcriptional activator that contains a typical acidic-hydrophobic domain that does not bind directly to DNA or RNA. The mechanism of action of this protein is more analogous to that of the transactivator proteins of adenovirus (E1a), human T-cell leukemia virus type 1 (Tax), and herpes simplex virus (VP-16) which interact with cellular proteins to activate transcription (90, 94, 173, 191). The visna virus Tat protein activates transcription via the AP-1 site most proximal to the TATA box (63). It also activates transcription of heterologous promoters (viral and cellular) that contain AP-1 sites. To dissect the functional domains of the visna virus Tat protein, the yeast Gal4 DNA binding domain was fused to the entire Tat protein or segments of the protein. An acidichydrophobic region at the amino terminus of the protein was found to contain the activation domain of the visna virus Tat protein (21). The function of the Tat protein is to increase the expression of viral mrna during the early phase of gene expression. This early phase of viral gene expression is characterized by the presence of spliced and unspliced viral RNAs in the nucleus but only fully spliced viral mrnas in the cytoplasm. This process results in the production of the regulatory proteins Tat, Rev, and Nef (39). A critical level of Rev protein is required to shift from the early phase of replication to the late phase of viral gene expression. This late phase is characterized by the

7 106 CLEMENTS AND ZINK CLIN. MICROBIOL. REV. FIG. 3. Spleen of sheep infected with OvLV strain 155. In situ hybridization demonstrates the localization of viral RNA in macrophages (arrowheads) around a splenic follicle. Hematoxylin staining was used. Magnification, 100. transport and translation of the structural and enzymatic genes from unspliced and partially spliced mrnas. The Rev protein facilitates the export of unspliced viral mrnas from the nucleus and the association of these viral RNAs with polyribosomes (3, 7). Rev proteins of lentiviruses share little amino acid homology but have common functions that can be attributed to two separate functional domains (147). One is a basic domain that acts as a nucleolar localization signal. Transiently expressed Rev protein of HIV-1, visna virus, and FIV localizes in the nucleolus of infected cells (127, 200). Recently, it has been shown in our laboratory that the Rev proteins of visna virus and CAEV localize in the nucleolus in infected primary cells in culture (181). The reason for Rev localizing in the nucleolus is still under investigation. The Rev protein functions by binding via its basic domain to highly structured RNA elements (the Rev-responsive element) present in the env genes of lentiviruses (32, 126, 158). Rev protein binds to RNA through the Rev-responsive element and facilitates the transport of unspliced and partially spliced viral mrna into the cytoplasm. The Rev protein may cause the utilization of an alternative RNA processing pathway that bypasses the splicing machinery and promotes the transport of the viral RNAs through the nucleolus. Cellular RNAs that are not processed via the cellular splicing machinery are found in the nucleolus. It is interesting to note that the Rev proteins of HIV-1, visna virus, and CAEV localize in the nucleolus regardless of whether viral RNAs are present in the cell. The expression of the unspliced viral RNA that encodes the structural proteins of the viral core (Gag proteins) and the enzymatic proteins of the pol gene, as well as that of the singly spliced mrna of the env gene, is dependent on the Rev protein. Late viral gene expression is initiated when there is sufficient Rev protein in the cell to facilitate the transport of the viral RNAs for these structural and enzymatic proteins. ROLE OF CELL TROPISM IN THE PATHOGENESIS OF UNGULATE LENTIVIRUSES The lentiviruses can be classified into two groups on the basis of whether they replicate mainly in macrophages or in both macrophages and lymphocytes. Replication in these cells of the immune system is responsible for specific clinical and pathological manifestations of disease. Cells of the monocyte/ macrophage lineage are the primary host cells for replication of OvLV and caprine lentivirus in vivo (16, 66, 108, 154, 165, 222, 224). Double-labeling experiments that use lectin histochemistry to identify macrophages and in situ hybridization or immunohistochemistry to detect viral RNA and proteins have confirmed that the vast majority of the infected cells in the central nervous system (CNS), lungs, spleen, lymph nodes, bone marrow, and joints of infected sheep and goats are macrophages (16, 66, 224). In HIV-infected humans and in SIVinfected macaques, virus replication occurs in both CD4 lymphocytes and macrophages, and replication in these two classes of cells is somewhat organ specific. For example, the majority of productively infected cells in the CNS and in the lungs of HIV- and SIV-infected individuals are of macrophage lineage, whereas infected lymphocytes are more commonly identified in lymphoid tissues such as the spleen and lymph nodes (45, 64, 107, 171, 184, 189, 206). These differences in cell tropism may be responsible for organ-specific manifestations of disease. In all of the lentiviruses, productive replication in macrophages is linked to cell maturation. Promonocytes and monocytes in the bone marrow and blood are infected, but viral replication in these cells is restricted and the virus remains in the form of proviral DNA. Upon maturation and differentiation to macrophages, either in tissue or in cell culture, cellular transcriptional factors that initiate viral transcription and sub-

8 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 107 FIG. 4. Lung of OvLV-infected sheep with diffuse interstitial pneumonia. There are thickened interalveolar septa (arrow) and numerous interstitial lymphocytic follicles (arrowhead). Hematoxylin staining was used. Magnification, 100. sequent production of viral proteins and virions are produced (5, 65, 118, 152). The lentiviruses that infect mainly macrophages, such as the OvLVs and CAEV, induce lesions in the same organs as the dual-tropic lentiviruses such as SIV and HIV, but the lesions are qualitatively different. In infected sheep and goats, productive virus replication in target tissues results in the development of an intense inflammatory response, with infiltration of macrophages and lymphocytes and even the production of focal lymphoid follicles in the affected tissues (Fig. 3). In contrast, in SIV and HIV the same organs are affected, with virus-infected macrophages prevalent, but there is much less of an inflammatory response. Infected sheep and goats frequently develop interstitial pneumonia characterized by thickened, often fibrotic, interalveolar septa and dense interstitial, perivascular, and peribronchial infiltrates of lymphocytes and macrophages (Fig. 4), (68, 69, 157). A large proportion of infected animals also develop an indurative mastitis with intense periductal and interstitial lymphocytic infiltrates (Fig. 5) (68, 122). Infected macrophages enter the milk from the inflamed mammary gland, and this provides the major mechanism of transmission of the virus from animal to animal (91, 135, 175). Some infected animals have arthritis, most often of the carpal joints but occasionally of the stifles or other joints. The affected joints have hyperplastic synovial membranes with large accumulations of lymphocytes, plasma cells, and macrophages in the subsynovial soft tissue (Fig. 6). In very advanced cases, there may be carpal bursitis, mineralization of the soft tissues of the joint, and erosion of the joint cartilage. Neurological disease occurs only rarely in infected sheep in North America, although during the Icelandic epizootic it was a relatively common manifestation in some flocks. Affected animals develop ataxia and paralysis. Pathological examination of the brain reveals intense perivascular inflammatory cuffs and diffuse infiltrates of lymphocytes and macrophages throughout the brain but most severe in the area around the lateral ventricles in the white matter (Fig. 7). The inflammation is frequently accompanied by demyelination, and myelin breakdown products can be demonstrated in macrophages within the neuropil (67, 167). This characteristic pattern of intense lymphoid infiltration and proliferation in the target organs is accompanied by marked hyperplasia of the associated lymph nodes, with an increase in the number of follicles and expansion of the paracortical zone (Fig. 8) (110). In many animals there is also generalized lymphoid hyperplasia that continues throughout the course of disease. This is in striking contrast to the immunodeficiency-causing lentiviruses, which induce early lymphoid hyperplasia that then gives way to lymphoid depletion during the later stages of disease. One mechanism for the lymphoid hyperplasia and inflammatory infiltrates that are seen in sheep infected with the OvLVs may be the upregulation of expression of major histocompatibility complex class II genes that occurs in infected macrophages (102). In addition, coculture of infected macrophages with lymphocytes elicits the secretion of a specific lentivirus-induced lymphokine called LV-IFN in the ovine-caprine system (109, 153, 223). In sheep, LV-IFN causes further upregulation of major histocompatibility complex class II expression while at the same time inhibiting the maturation of the host macrophage, thus slowing virus replication. LV-IFN is thought to maintain continual expression of major histocompatibility complex class II antigen and viral antigen in the infected macrophage, while virion assembly is inhibited, thus setting the stage for a severe inflammatory reaction. This hypothesis was confirmed by infusing LV-IFN into the brains of infected sheep and demonstrating a significant increase in the severity of encephalitis (220). Because of the lymphocyte tropism of many of the other FIG. 5. Mammary gland from an OvLV-infected sheep with numerous periductal and interstitial lymphoid aggregates (arrowhead). d, duct. Hematoxylin staining was used. Magnification, 200.

9 108 CLEMENTS AND ZINK CLIN. MICROBIOL. REV. FIG. 6. Synovial tissue from a sheep infected with OvLV strain 155. There is villous hyperplasia of synovium with accumulations of lymphocytes, plasma cells, and macrophages (arrow). Hematoxylin staining was used. Magnification, 200. lentiviruses, it was of interest to know whether the lentiviruses of sheep and goats could replicate in these cells. Viral RNA has been detected in small cells that morphologically resemble lymphocytes in the brains, lungs, spleens, lymph nodes, and small intestines of naturally infected goats (224). To further investigate the possibility of lymphocyte infection in vivo, we removed bone marrow cells from lentivirus-infected sheep and separated the cells according to density by countercurrent centrifugation (elutriation). Approximately 1% of the cells in the fraction containing only small and medium lymphocytes were positive for viral RNA by in situ hybridization. Efforts to rescue virus from cultured lymphocytes, however, have had conflicting FIG. 7. Brain of a sheep naturally infected with OvLV. In this section of periventicular white matter, there is an intense infiltrate of macrophages and lymphocytes diffusely throughout the neuropil and in large perivascular cuffs (arrows). Hematoxylin staining was used. Magnification, 100.

10 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 109 FIG. 8. Follicular hyperplasia of the lymph node of an OvLV-infected sheep. Multiple active follicles are present in the cortex (arrowheads). Hematoxylin staining was used. Magnification, results (78, 166). Taken together, these data suggest that small numbers of lymphocytes may be infected but that virus replication in these cells does not occur to a great extent. Certainly, the clinical and pathological evidence suggests that if lymphocytes are infected, the infection does not result in the gross alterations of immune function typical of the immunodeficiency-causing lentiviruses. Recent studies in this laboratory on the pathogenesis of neurological disease in lentivirus-infected sheep have demonstrated that a number of different strains of OvLV replicate productively in cultured CNS endothelial cells. Inoculated cultures develop syncytia within 2 days (Fig. 9), and virus replication in these cells is confirmed by PCR, in situ hybridization, and immunohistochemistry on the inoculated cells and by the demonstration of rising titers of virus in the supernatants. Electron microscopic examination of the inoculated cells demonstrated virus budding into subplasmalemmal vacuoles in the endothelial cells. Brain microvascular endothelial cells cultured from two sheep from an infected flock developed syncytia and were positive for viral DNA by PCR, indicating that infection of sheep CNS endothelial cells is also an in vivo phenomenon. Infection of endothelial cells may have a number of pathogenetic consequences. In addition to providing a possible route for virus entry to the CNS, endothelial infection may result in alterations to the blood-brain barrier and thus contribute to further virus entry, augmentation of virus load in the CNS, and the development of clinical signs of neurological disease. There is evidence that a number of other cells may be infected by the small ruminant lentiviruses. Viral RNA has been detected in epithelial cells of the thyroids, kidneys, and small intestines of goats with CAEV-induced leukoencephalomyelitis (224). In addition, a comprehensive study of the tropism of visna virus in the CNS of Icelandic sheep demonstrated viral FIG. 9. Cultured sheep microvessel endothelial cells inoculated with visna virus develop syncytia (arrowheads) after 3 days, indicative of productive virus replication. Phase microscopy was used. Magnification, 400.

11 110 CLEMENTS AND ZINK CLIN. MICROBIOL. REV. FIG. 10. Spinal cord from a macaque inoculated with a neurovirulent variant of SIV mac. Macrophages are the predominant cell type in perivascular cuffs (arrows), and there is a diffuse increase in the cellularity of the neuropil. Hematoxylin staining was used. Magnification, 200. antigen in a wide variety of cells in the CNS, including epithelial cells and fibroblasts of the choroid plexus and cells that morphologically resembled endothelial cells, pericytes, lymphocytes, and plasma cells (67). Thus, it is becoming evident that although the macrophage is the main cell for productive virus replication, the sheep and goat lentiviruses do infect a broader range of cells than was first thought. The replication of virus in these additional cell types raises the possibility that the virus may alter the function of these organs and contribute to disease pathogenesis in sheep and goats in ways that have not yet been recognized. ROLE OF CELL TROPISM IN SIV PATHOGENESIS Unlike the OvLVs, the SIVs replicate productively in both CD4 monocytes/macrophages and lymphocytes. While some strains replicate to high titers in lymphocytes and only minimally in macrophages, other strains are able to replicate equally well in both cell types. Replication in these two cell types is thought to be responsible for different manifestations of disease. Virus replication in cells of monocyte/macrophage lineage results in primary disease manifestations in the CNS and lung, whereas virus replication in lymphocytes is responsible for immunosuppression and the development of opportunistic infections. SIV-infected macaques (and HIV-infected humans) initially develop lymphoid hyperplasia, but unlike in sheep and goats, this hyperplasia eventually gives way to lymphoid depletion during the later stage of disease. Lesions in the CNS of infected macaques are different from those of sheep and goats with respect to their location and their character. In visna virus-infected sheep, lesions are located in the lateral ventricles and characterized by an intense cellular inflammatory response. The lesions of macaques are distributed throughout the brain and spinal cord and are most intense in the white matter of basal ganglia and thalamus, with minimal cellular infiltration. Typically, the CNS of infected macaques contains numerous perivascular cuffs consisting mainly of macrophages with some lymphocytes, with variable numbers of multinucleated giant cells (Fig. 10). Focal microglial nodules and multinucleated giant cells can also be seen throughout the neuropil. Like infected sheep and goats, SIV-infected macaques develop interstitial pneumonia, but the predominant histological feature in macaques is the presence of multinucleated giant cells that contain viral gene products (Fig. 11). Perhaps the most significant difference in the manifestations of these two groups of lentiviruses is the presence of secondary opportunistic infections in HIV- and SIV-infected individuals. Toxoplasmosis, cytomegalovirus, and aspergillosis are commonly seen in the CNS, whereas Pneumocystis carinii is found in the lung. The development of neurological disease in SIV-infected macaques has been shown to be associated with macrophagetropic strains of SIV. In one study, macaques were inoculated with a molecularly cloned, strictly lymphocyte-tropic strain of SIV, SIV mac 239. After several months passed to allow time for the development of variants, virus was isolated from the brain and bone marrow of this animal and inoculated intracerebrally into another macaque. After this procedure had been repeated three times, the virus had a broadened tropism and was able to replicate to high titers in macrophages. Animals infected with the macrophage-tropic virus developed typical neurological disease characterized by the presence of perivascular macrophage-rich cuffs and multifocal microglial nodules throughout the parenchyma (Fig. 10) (184). Coincidentally, these animals also developed interstitial pneumonia with abundant intra-alveolar infiltrates of macrophages and giant cells (Fig. 11). Double labeling by lectin histochemistry to identify macrophages and in situ hybridization to detect viral RNA showed that the only

12 VOL. 9, 1996 PATHOGENESIS OF ANIMAL LENTIVIRUS INFECTIONS 111 FIG. 11. Lung from a macaque inoculated with a macrophage-tropic strain of SIV mac. There is interstitial pneumonia with thickening of interalveolar septa and large numbers of multinucleated giant cells throughout the interstitium (arrows). Hematoxylin staining was used. Magnification, 200. infected cells in these tissues were macrophages. In another study, animals were inoculated with the same lymphocytetropic strain, SIV mac 239, and examined after 18 months. Two of five inoculated animals developed neurological disease. Virus isolated from these two animals was shown to be macrophage-tropic, suggesting that a shift from lymphocyte to macrophage tropism had occurred prior to the development of neurological disease in these animals (15). For reasons that are still not completely understood, replication of the virus in lymphocytes causes a loss of CD4 lymphocytes in lymphoid tissues, and this is reflected in a decrease in the absolute number of CD4 lymphocytes in the peripheral blood (11). The loss of CD4 cells and other, more global alterations in the hosts immune responses contribute to the immunosuppression and hence the life-threatening secondary opportunistic infections seen in individuals infected with the lentiviruses from this group. SIV-infected macaques develop the same spectrum of opportunistic infections as human AIDS patients, including P. carinii pneumonia, cytomegalovirus pneumonia and encephalitis, and Candida esophagitis. Like the lentiviruses of sheep, SIV has been shown to replicate in CNS endothelial cells both in vivo and in vitro. SIV RNA was detected in endothelial cells by double immunohistochemistry and in situ hybridization and by reverse transcriptase-in situ PCR in macaques inoculated with neurovirulent strains of SIV but not non-neurovirulent strains (128). Further studies with primary cultures of macaque brain endothelial cells showed that four neurovirulent strains of SIV replicated productively in the cells, whereas three strains that did not cause neurological disease did not replicate in these cells (Table 1). Virus entry occurred via a CD4-independent mechanism. These in vivo and in vitro findings suggest that replication of the virus in endothelial cells plays a role in the development of SIV-induced neurological disease. Infection of endothelial cells may increase virus entry to the CNS, either directly or as a result of virus-induced alterations of the bloodbrain barrier. Alterations in the integrity of the blood-brain barrier may, in turn, contribute to the development of dementia and encephalitis. Several strains of HIV also have been shown to replicate in primary cultures of human CNS endothelial cells (143, 169), and replication in CNS endothelial cells is a common pathogenetic mechanism among other retroviruses (89, 161). The finding that the lentiviruses of humans, macaques, and sheep replicate in CNS endothelial cells suggests a pathogenetic mechanism common to the lentiviruses. A major conclusion to be drawn from these studies is that the molecular basis for neurological disease may lie not only in those viral sequences that confer upon the virus the ability to replicate in macrophages but also in sequences that mediate tropism for specific target cells such as brain endothelial cells. A virus strain with a specific tissue tropism is most probably selected by replication in that tissue from among the many TABLE 1. Relationship between cell tropism and neurovirulence of SIV strains Virus strain Tropism a Lymphocytes Macrophages Brain Neurovirulence b endothelial cells SIV mac 239 SIV mac 239/17E-Br SIV mac 239/17E-C1 SIV mac 23917E-C1 nef SIV mac 239/17E-Fr SIV mac 251 SIV B670 a, productive replication;, no virus replication. b, presence of lesions in CNS;, absence of lesions in CNS.