Feline Pit2 functions as a receptor for subgroup B feline leukemia viruses.

Different subgroups of feline leukemia virus (FeLV) use different host cell receptors for entry. Subgroup A FeLV (FeLV-A) is the virus that is transmitted from cat to cat, suggesting that cells expressing the FeLV-A receptor are important targets at the earliest stages of infection. FeLV-B evolves from FeLV-A in the infected cat through acquisition of cellular sequences that are related to the FeLV envelope gene. FeLV-Bs have been shown to infect cells using the Pit1 receptor, and some variants can infect cells at a lower efficiency using Pit2. Because these observations were made using receptor proteins of human or rodent origin, the role that Pit1 and Pit2 may play in FeLV-B replication in the cat is unclear. In this study, the feline Pit receptors were cloned and tested for their ability to act as receptors for different FeLV-Bs. Some FeLV-Bs infected cells expressing feline Pit2 and feline Pit1 with equal high efficiency. Variable region A (VRA) in the putative receptor-binding domain (RBD) was a critical determinant for both feline Pit1 and feline Pit2 binding, although other domains in the RBD appear to influence how efficiently the FeLV-B surface unit can bind to feline Pit2 and promote entry via this receptor. An arginine residue at position 73 in VRA was found to be important for envelope binding to feline Pit2 but not feline Pit1. Interestingly, this arginine is not found in endogenous FeLV sequences or in recombinant viruses recovered from feline cells infected with FeLV-A. Thus, while FeLV-Bs that are able to use feline Pit2 can evolve by recombination with endogenous sequences, a subsequent point mutation during reverse transcription may be needed to generate a virus that can efficiently enter the cells using the feline Pit2 as its receptor. These studies suggest that cells expressing the feline Pit2 protein are likely to be targets for FeLV-B infection in the cat.

Specificity in receptor usage by T-cell-tropic feline leukemia viruses: implications for the in vivo tropism of immunodeficiency-inducing variants.

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828-1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.

Receptors and entry cofactors for retroviruses include single and multiple transmembrane-spanning proteins as well as newly described glycophosphatidylinositol-anchored and secreted proteins.

In the past few years, many retrovirus receptors, coreceptors, and cofactors have been identified. These molecules are important for some aspects of viral entry, although in some cases it remains to be determined whether they are required for binding or postbinding stages in entry, such as fusion. There are certain common features to the molecules that many retroviruses use to gain entry into the cell. For example, the receptors for most mammalian oncoretroviruses are multiple membrane-spanning transport proteins. However, avian retroviruses use single-pass membrane proteins, and a sheep retrovirus uses a glycosylphosphatidylinositol-anchored molecule as its receptor. For some retroviruses, particularly the lentiviruses, two cell surface molecules are required for efficient entry. More recently, a soluble protein that is required for viral entry has been identified for a feline oncoretrovirus. In this review, we will focus on the various strategies used by mammalian retroviruses to gain entry into the cell. The choice of receptors will also be discussed in light of pressures that drive viral evolution and persistence.

Identification of envelope determinants of feline leukemia virus subgroup B that permit infection and gene transfer to cells expressing human Pit1 or Pit2.

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.

Selection forces and constraints on retroviral sequence variation.

All retroviruses possess a highly error-prone reverse transcriptase, but the extent of the consequent sequence diversity and the rate of evolution differ greatly among retroviruses. Because of the high mutability of retroviruses, it is not the generation of new viral variants that limits the extent of diversity and the rate of evolution of retroviruses, but rather the selection forces that act on these variants. Here, we suggest that two selection forces--the immune response and the limited availability of appropriate target cells during transmission and persistence--are chiefly responsible for the observed sequence diversity in untreated retroviral infections. We illustrate these aspects of positive selection by reference to specific lentiviruses [human and simian immunodeficiency viruses (HIV and SIV)] and oncoviruses [feline leukemia virus (FeLV) and human T cell leukemia virus (HTLV)] that differ in their extent of variation and in disease outcomes.

Use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and T tropism: evidence for distinct levels of restriction in primary macrophages and a T-cell line.

Cell tropism of human and simian immunodeficiency viruses (HIV and SIV, respectively) is governed in part by interactions between the viral envelope protein and the cellular receptors. However, there is evidence that envelope-host cell interactions also affect postentry steps in viral replication. We used a helper-free replication-defective SIV macaque (SIVmac)-based retroviral vector carrying the enhanced jellyfish green fluorescent protein inserted into the nef region (V1EGFP) to examine SIV tropism in a single cycle of infection. Vector stocks containing envelope proteins from three different SIVmac clones, namely, SIVmac239 (T-lymphocyte tropic [T-tropic]), SIVmac316 (macrophage tropic [M-tropic]), and SIVmac1A11 (dualtropic), were tested. SIVmac239 replicates efficiently in many human T-cell lines, but it does not efficiently infect primary rhesus macrophages. Conversely, SIVmac316 efficiently infects primary macrophages, but it does not replicate in Molt4-Clone8 (M4C8) T cells. SIVmac1A11 replicates efficiently in both cell types. When primary macrophages were infected with V1EGFP pseudotyped by SIVmac316 or SIVmac1A11 envelopes, the infection was substantially (ca. 200- to 300-fold) more efficient than for the SIVmac239 pseudotype. Thus, in primary macrophages, a major component of M versus T tropism involves relatively early events in the infection cycle. Quantitative PCR studies indicated that synthesis and transport of vector DNA into the nucleus were similar for macrophages infected with the clone 239 and 316 pseudotypes, suggesting that the restriction for SIVmac239 infection is after reverse transcription and nuclear import of viral DNA. When the same vector pseudotypes were used to infect M4C8 cells, they all showed approximately equivalent infectivities, even though replication-competent SIVmac316 does not continue to replicate in these cells. Therefore, in M4C8 cells, restriction involves a late step in the infection cycle (after proviral integration and expression). Thus, depending on the cell type infected, envelope-dependent cell interactions that govern SIV M and T tropism may involve different steps in infection.

Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein.

We previously reported the isolation from a thymic tumor of a feline leukemia virus that had transduced a fragment of the Notch2 gene. Here we present evidence that a nuclear form of Notch2 corresponding to the biologically active intracellular domain (N2ICD) is expressed from this recombinant retrovirus through internal ribosome entry. Internal ribosome entry sites (IRESs) are RNA structural motifs that allow 5' cap-independent recruitment of ribosomal subunits to mRNAs. The Notch2 IRES maps exclusively to Notch2 sequences that correspond to the coding region of the cellular gene. Therefore, these studies not only provide insights into aberrant Notch2 expression in tumors, but they may also inform our understanding of N2ICD generation in the cellular context.

Feline leukemia virus envelope sequences that affect T-cell tropism and syncytium formation are not part of known receptor-binding domains.

The envelope protein is a primary pathogenic determinant for T-cell-tropic feline leukemia virus (FeLV) variants, the best studied of which is the immunodeficiency-inducing virus, 61C. We have previously demonstrated that T-cell-tropic, cytopathic, and syncytium-inducing viruses evolve in cats infected with a relatively avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T variant, which encoded scattered single-amino-acid changes throughout the envelope as well as a 4-amino-acid insertion in the C-terminal half of the surface unit (SU) of envelope, was sufficient to confer the T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser, S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol. 72:2686-2696, 1998). In the present study, we examined the role of the 4-amino-acid insertion in determining viral replication and tropism of FeLV-81T. The 4-amino-acid insertion was found to be functionally equivalent to a 6-amino-acid insertion at an identical location in the 61C variant. However, viruses expressing a chimeric 61E/81T SU, containing the insertion together with the N terminus of 61E SU, were found to be replication defective and were impaired in the processing of the envelope precursor into the functional SU and transmembrane (TM) proteins. In approximately 10% of cultured feline T cells (3201) transfected with the 61E/81T envelope chimeras and maintained over time, replication-competent tissue culture-adapted variants were isolated. Compensatory mutations in the SU of the tissue culture-adapted viruses were identified at positions 7 and 375, and each was shown to restore envelope protein processing when combined with the C-terminal 81T insertion. Unexpectedly, these viruses displayed different phenotypes in feline T cells: the virus with a change from glutamine to proline at position 7 acquired a T-cell-tropic, cytopathic phenotype, whereas the virus with a change from valine to leucine at position 375 had slower replication kinetics and caused no cytopathic effects. Given the differences in the replication properties of these viruses, it is noteworthy that the insertion as well as the two single-amino-acid changes all occur outside of predicted FeLV receptor-binding domains.

Identification of a cellular cofactor required for infection by feline leukemia virus.

Retroviral infection involves continued genetic variation, leading to phenotypic and immunological selection for more fit virus variants in the host. For retroviruses that cause immunodeficiency, pathogenesis is linked to the emergence of T cell-tropic, cytopathic viruses. Here we show that an immunodeficiency-inducing, T cell-tropic feline leukemia virus (FeLV) has evolved such that it cannot infect cells unless both a classic multiple membrane-spanning receptor molecule (Pit1) and a second coreceptor or entry factor are present. This second receptor component, which we call FeLIX, was identified as an endogenously expressed protein that is similar to a portion of the FeLV envelope protein. This cellular protein can function either as a transmembrane protein or as a soluble component to facilitate infection.

Cloning of the cellular receptor for feline leukemia virus subgroup C (FeLV-C), a retrovirus that induces red cell aplasia.

Feline leukemia virus-C (FeLV-C) causes red cell aplasia in cats, likely through its interaction with its cell surface receptor. We identified this receptor by the functional screening of a library of complementary DNAs (cDNA) from feline T cells. The library, which was cloned into a retroviral vector, was introduced into FeLV-C-resistant murine (NIH 3T3) cells. The gene conferring susceptibility to FeLV-C was isolated and reintroduced into the same cell type, as well as into FeLV-C-resistant rat (NRK 52E) cells, to verify its role in viral infection. The receptor cDNA is predicted to encode a protein of 560 amino acids with 12 membrane-spanning domains, termed FLVCR. FLVCR has significant amino acid sequence homology with members of the major facilitator superfamily and especially D-glucarate transporters described in bacteria and in C. elegans. As FeLV-C impairs the in vivo differentiation of burst-forming unit-erythroid to colony-forming unit-erythroid, we hypothesize that this transporter system could have an essential role in early erythropoiesis. In further studies, a 6-kb fragment of the human FLVCR gene was amplified by polymerase chain reaction from genomic DNA, using homologous cDNA sequences identified in the human Expressed Sequence Tags database. By radiation hybrid mapping, the human gene was localized to a 0.5-centiMorgan region on the long arm of chromosome 1 at q31.3.

Gender differences in HIV-1 diversity at time of infection.

To develop an HIV-1 vaccine with global efficacy, it is important to identify and characterize the viruses that are transmitted, particularly to individuals living in areas of high incidence. Several studies have shown that virus from the blood of acutely infected adults was homogeneous, even when the virus population in the index case was genetically diverse. In contrast to those results with mainly male cohorts in America and Europe, in several cases a heterogeneous virus population has been found early in infection in women in Africa. Thus, we more closely compared the diversity of transmitted HIV-1 in men and women who became infected through heterosexual contact. We found that women from Kenya were often infected by multiple virus variants, whereas men from Kenya were not. Moreover, a heterogeneous virus was present in the women before their seroconversion, and in each woman it was derived from a single index case, indicating that diversity was most likely to be the result of transmission of multiple variants. Our data indicate that there are important differences in the transmitted virus populations in women and men, even when cohorts from the same geographic region who are infected with the same subtypes of HIV-1 are compared.

Lymphokines modulate the growth and survival of thymic tumor cells containing a novel feline leukemia virus/Notch2 variant.

Tumorigenesis occurs through a multistep process initiated by genetic lesions and facilitated by endogenous and external growth/survival signals. In many malignancies, specific oncogenic mutations correlate with phenotypic characteristics, inferring lineage-specific pathogenic mechanisms. To characterize these relationships in a unique feline tumor, we studied primary cells and two-cell lines independently-derived from a thymic lymphoma that contained and actively expressed a novel feline leukemia virus (FeLV) recombinant with transduced host Notch2 sequences. All three tumor cell populations contained similar FeLV/Notch2 proviral variants and phenotypically resembled mature thymocytes. Multiple Notch2 transcripts were expressed in the cell lines, including species that correspond to viral genomes and spliced subgenomic viral mRNA. Tumor cell line FeLV/Notch2 virus was packaged into virions; however, the variant was not efficiently transmitted to feline cells in vitro. Primary tumor cells constitutively expressed mRNA for interleukin-4 (IL-4), IL-6 and the p40 subunit of IL-12. Lymphokine mRNA was not detected in established tumor cell lines nor was T-cell growth-promoting activity found in culture supernatants. Exogenous IL-4 enhanced primary tumor cell survival, but inhibited proliferation of the cell lines. Interleukin-4 abrogated hydrocortisone-induced apoptosis in all three populations and had divergent effects on cell line clonogenic colony formation. Exogenous IL-7 and, to a lesser degree, IL-6 also had variable positive effects on the growth and viability of the tumor cell populations. Collectively, these data suggest that thymocytes are susceptible to the transforming potential of dysregulated Notch2 and that thymopoietic factors could, through overlapping and distinct mechanisms, promote the survival and outgrowth of FeLV/Notch2-containing neoplastic cells.

Limited breadth of the protective immunity elicited by simian immunodeficiency virus SIVmne gp160 vaccines in a combination immunization regimen.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

An investigation of genital ulcers in Jackson, Mississippi, with use of a multiplex polymerase chain reaction assay: high prevalence of chancroid and human immunodeficiency virus infection.

In 1994, an apparent outbreak of atypical genital ulcers was noted by clinicians at the sexually transmitted disease clinic in Jackson, Mississippi. Of 143 patients with ulcers tested with a multiplex polymerase chain reaction (PCR) assay, 56 (39%) were positive for Haemophilus ducreyi, 44 (31%) for herpes simplex virus, and 27 (19%) for Treponema pallidum; 12 (8%) were positive for > 1 organism. Of 136 patients tested for human immunodeficiency virus (HIV) by serology, 14 (10%) were HIV-seropositive, compared with none of 200 patients without ulcers (P < .001). HIV-1 DNA was detected by PCR in ulcers of 6 (50%) of 12 HIV-positive patients. Multivariate analysis indicated that men with chancroid were significantly more likely than male patients without ulcers to report sex with a crack cocaine user, exchange of money or drugs for sex, and multiple sex partners. The strong association between genital ulcers and HIV infection in this population highlights the urgency of preventing genital ulcers in the southern United States.

Evolution of envelope sequences from the genital tract and peripheral blood of women infected with clade A human immunodeficiency virus type 1.

The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.

In vivo evolution of a novel, syncytium-inducing and cytopathic feline leukemia virus variant.

Studies of feline leukemia virus (FeLV) have illustrated the importance of the genotype of the infecting virus in determining disease outcome. In FeLV infections, as in other retroviral infections, it is less clear how virus variants that evolve from the transmitted virus affect pathogenesis. We previously reported an analysis of the genotypic changes that occur in the viral envelope gene (env) in cats infected with a prototype transmissible FeLV clone, 61E (J. Rohn, M. Linenberger, E. Hoover, and J. Overbaugh, J. Virol. 68:2458-2467, 1994). In one cat, each variant (81T) had evolved, in addition to scattered amino acid changes, a four-amino-acid insertion with respect to 61E. This insertion was located at the same site in the extracellular envelope glycoprotein where the immunodeficiency-inducing molecular clone 61C possesses a six-amino-acid insertion critical for its pathogenic phenotype, although the sequences of the insertions were distinct. To determine whether acquisition of the four-amino-acid insertion was associated with a change in the replication or cytopathic properties of the virus, we constructed chimeras encoding 81T env genes in a 61E background. One representative chimeric virus, EET(TE)-109, was highly cytopathic despite the fact that it replicated with delayed kinetics in the feline T-cell line 3201 compared to the parental 61E virus. The phenotype of this virus was also novel compared to other FeLVs, including both the parental virus 61E and the immunodeficiency-inducing variant 61C, because infection of T cells was associated with syncytium formation. Moreover, in single-cycle infection assays, the 81T-109 envelope demonstrated receptor usage properties distinct from those of both 61E and 61C envelope. Thus, these studies demonstrate the evolution of a novel T-cell cytopathic and syncytium-inducing FeLV in the host. The 81T virus will be valuable for dissecting the mechanism of T-cell killing by cytopathic variants in the FeLV model.

The host range and interference properties of two closely related feline leukemia variants suggest that they use distinct receptors.

The proviral clones 61E and 61C represent two closely related variants of feline leukemia virus (FeLV) that exhibit significant differences in their biological and pathogenic properties. The major pathogenic determinant has been mapped to the extracellular envelope glycoprotein (Env-SU), but the mechanism by which envelope differences influence pathogenesis is not well understood. Moreover, it is unclear whether these viruses infect the same target cells and/or enter cells using the same receptor. In the present study, we exploited a recently developed single cycle infection assay to examine the host range and interference properties of 61E and 61C FeLVs and found that these two FeLV variants differ significantly in their host ranges and receptor usages. FeLV-61C was found to be an ecotropic virus; the entry of viruses bearing a 61C envelope protein (Env-SU) into cell lines was limited to feline T-cells and feline fibroblasts. In contrast, the host range of 61E includes, in addition to all feline cells examined, some canine, murine, and human cell lines. Feline fibroblast and feline T-cells that expressed 61E envelope were resistant to infection with a virus bearing a 61E Env-SU, whereas these same cells were susceptible to infection by an otherwise similar virus pseudotyped with the 61C Env-SU. This pattern of interference was observed in cells expressing 61E envelope alone, in the absence of other FeLV gene products, demonstrating that interference was mediated specifically by Env-SU. Fibroblast cells chronically infected with a 61C virus were partially resistant to infection with a virus having a 61C Env-SU, but were not resistant to infection by a virus having a 61E Env-SU. On the basis of the current understanding of virus-receptor interactions, the lack of interference between 61E and 61C under conditions where there is significant homologous interference, combined with the differences in their host cell range, leads us to conclude that 61E and 61C use two distinct primary cellular receptors for entry.

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.

Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies.

During progression to AIDS in simian immunodeficiency virus (SIV) Mne-infected macaques, viral variants are selected that encode sequences with serine and threonine changes in variable region 1 (V1) of the surface component of the viral envelope protein (Env-SU). Because these serine and threonine amino acid changes are characteristic of sites for O-linked and N-linked glycosylation, we examined whether they were targets for modification by carbohydrates. For this purpose, we used several biochemical methods for analyzing the Env-SU protein encoded by chimeras of SIVMneCL8 and envelope sequences cloned from an SIVMneCL8-infected Macaca nemestrina during clinical latency and just after the onset of AIDS. The addition of an N-linked glycan was demonstrated by changes in the electrophoretic mobility of Env-SU, and this was verified by specific glycanase digestions and a detailed analysis of the molecular mass of partially purified Env-SU by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Molecular mass calculations by MALDI-TOF MS also demonstrated an increased mass, from 102.3 to 103.5 kDa, associated with serine and threonine residues predicted to be O-linked glycosylation sites. Together, these data provide the first direct evidence that the carbohydrate profile of Env-SU is distinct in SIV variants that evolve during infection of the host. Moreover, our studies show that these changes in glycosylation in V1 were directly associated with changes in antigenicity. Specifically, serine and threonine changes in V1 allowed the virus to escape neutralization by macaque sera that contained antibodies that could neutralize the parental virus, SIVMneCL8. The escape from antibody recognition appeared to be influenced by either O-linked or N-linked carbohydrate additions in V1. Moreover, when glycine residues were engineered at the positions where serine and threonine changes evolve in V1 of SIVMneCL8, there was no change in antigenicity compared to SIVMneCL8. This suggests that the amino acids in V1 are not part of the linear epitope recognized by neutralizing antibody. More likely, V1-associated carbohydrates mask the major neutralizing epitope of SIV. These experiments indicate that the selection of novel glycosylation sites in the V1 region of envelope during the course of disease is driven by humoral immune responses.