Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques.

Genetic diversity is a hallmark of the human immunodeficiency virus (HIV) genome, but the role of distinct HIV variants in the development of AIDS is unclear. Envelope (env) is the most highly variable gene in HIV as well as in other retroviruses. We have previously demonstrated that variation in simian immunodeficiency virus (SIV) env is primarily localized in two regions (V1 and V4) during progression to simian AIDS. To determine whether there is a common genotype that evolves as AIDS develops, a total of 160 SIV env genes isolated directly from the tissue DNAs of four macaques infected with cloned virus were compared. Common amino acid sequence changes were identified within V1, V4, and, in the late stages of disease, near V3. At several positions, the same amino acid change was seen frequently in the variant genomes from all four animals. As AIDS developed, the majority of viruses evolved an extended sequence in V1 that was rich in serine and threonine residues and shared similarity with proteins modified by O-linked glycosylation. Several of the predominant common sequence changes in V1 and V4 created new sites for N-linked glycosylation. Thus, common features of the SIV variants that evolve during progression to AIDS are motifs that potentially allow for structural and functional changes in the env protein as a result of carbohydrate addition.     

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.     

Unique pattern of convergent envelope evolution in simian immunodeficiency virus-infected rapid progressor macaques: association with CD4-independent usage of CCR5

The rate of disease development in simian immunodeficiency virus (SIV) infection of macaques varies considerably among individual macaques. While the majority of macaques inoculated with pathogenic SIV develop AIDS within a period of 1 to 2 years, a minority exhibit a rapid disease course characterized by absence or transience of humoral and cellular immune responses and high levels of virus replication with widespread dissemination of SIV in macrophages and multinucleated giant cells. The goal of this study was to examine viral evolution in three SIVsmE543-3-inoculated rapid progressors to determine the contribution of viral evolution to the development of rapid disease and the effect of the absence of immune pressure upon viral evolution. PCR was used to amplify and clone the entire SIV genome from tissues collected at necropsy, and the course of viral evolution was assessed by env sequences cloned from sequential plasma samples of one rapid progressor (RP) macaque. The majority of sequence changes in RP macaques occurred in the envelope gene. Substitutions were observed in all three animals at specific conserved residues in envelope, including loss of a glycosylation site in V1/V2, a D-to-N/V substitution in a highly conserved GDPE motif, and a P-to-V/H/T substitution in the V3 loop analog. A cell-cell fusion assay revealed that representative env clones utilized CCR5 as a coreceptor, independent of CD4. The selection of specific substitutions in envelope in RP macaques suggests novel selection pressures on virus in such animals and suggests that viral variants that evolve in these animals may play a role in disease progression.     

Replication and persistence of simian immunodeficiency virus variants after passage in macaque lymphocytes and established human cell lines.

In lentivirus infections, there are typically few cells in the host that harbor the provirus. For this reason, molecular clones of human and simian immunodeficiency viruses (HIV and SIV) are generally derived after passage and amplification of the virus in cell culture. To determine whether SIV variants that persist in culture are similar to the variants that predominate in the host, we examined the proviral sequence of the SIV envelope (env) gene before and after cocultivation of lymphocytes from a macaque with AIDS with naive macaque lymphocytes or human cell lines. Many of the predominant variants in the monkey replicated and persisted in macaque lymphocytes and CEMx174 cells in culture, but a more limited population of variants replicated in C8166 cells. Passage of virus, harvested after 4 weeks of coculture, onto naive cells further demonstrated that the majority of proviruses detected by polymerase chain reaction were also viral variants that were expressed and packaged into infectious virions.     

Correlation between env V1/V2 region diversification and neutralizing antibodies during primary infection by simian immunodeficiency virus sm in rhesus macaques

Evolution of the domain encoding the V1/V2 variable region of the simian immunodeficiency virus sm (SIVsm) envelope (env) gene was analyzed in relation to route of virus challenge, virus load, and neutralizing antibody (NAb) titers during primary infection of rhesus macaques with the pathogenic SIVsmE660 isolate. In this model system animals are initially infected with multiple viruses as evidenced by the presence of multiple V1/V2 genotypic variants that could be resolved by using a heteroduplex tracking assay (HTA). Overlapping subsets of the multiple variants were established in each animal. There was no selection for the establishment of specific variants in comparing intravenous- and intrarectal-challenged macaques at week 2 postinfection, suggesting that no genotypic selection occurred at the mucosal surface. There was an initial period of significant stability of the V1/V2 variants. Macaques challenged intravenously displayed subsequent V1/V2 diversification significantly earlier than macaques challenged intrarectally and well past the initial resolution of viremia. The time when SIVsmE660-specific NAbs reached a threshold titer of 100 was significantly correlated with the timing of V1/V2 diversification, even though antibodies to the Env protein could be detected much earlier. The time when NAbs reached a titer of 400 was significantly correlated with virus load late in infection. These results show that the route of infection affects the timing of V1/V2 diversification and that this diversification is correlated with the maturation of a specific NAb response. However, prior immunization capable of priming an anamnestic Env antibody response did not accelerate V1/V2 diversification. This result suggests that diversification of the SIV env V1/V2 region is the result of a type-specific antibody response.     

A dominant role for CD8+-T-lymphocyte selection in simian immunodeficiency virus sequence variation

CD8(+) T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope.     

Extensive envelope heterogeneity of simian immunodeficiency virus in tissues from infected macaques.

The extent of virus genetic variation within tissues and peripheral blood mononuclear cells (PBMC) from two simian immunodeficiency virus (SIV)-infected macaques was analyzed. The products of PCR amplification of two regions, region 1 (SIV V1 region) and region 2 (region corresponding to the human immunodeficiency virus V3 cysteine loop and part of the C3 region immediately downstream), of the SIV envelope were examined for single-stranded conformation polymorphism followed by sequence analysis of selected clones. The V1 region of the SIV envelope of viruses present within lymphoid tissues displayed extensive heterogeneity, while viral populations within the PBMC and brain appeared to be less variable. Region 2 heterogeneity in both animals was generally confined to three residues in a tissue-specific manner. In addition, virus from the brains of both animals appeared to be distinct compared with viruses present in other tissues and PBMC of the same animal, both in the pattern of PCR-single-stranded conformation polymorphism SCP and in the sequence of region 2. These studies revealed that the tissues of SIV-infected macaques were a reservoir for viral variants distinct from those seen in PBMC.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus env proteins possess a functionally conserved assembly domain.

The envelope (env) glycoproteins of human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) form dimers shortly after synthesis. Analysis of the simian immunodeficiency virus (SIV) env protein expressed by a recombinant vaccinia virus revealed that it, too, forms stable homodimers. When the HIV-1 and SIV env proteins or the HIV-1 and HIV-2 env proteins were coexpressed in the same cells, heterodimers were formed. Thus, the env proteins of HIV-1, HIV-2, and SIV possess a functionally conserved domain involved in subunit-subunit recognition and assembly that likely involves the ectodomain of gp41.     

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.     

Removal of N-linked glycosylation sites in the V1 region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.     

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression

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

Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with simian AIDS.

One rhesus macaque displayed severe encephalomyelitis and another displayed severe enterocolitis following infection with molecularly cloned simian immunodeficiency virus (SIV) strain SIVmac239. Little or no free anti-SIV antibody developed in these two macaques, and they died relatively quickly (4 to 6 months) after infection. Manifestation of the tissue-specific disease in these macaques was associated with the emergence of variants with high replicative capacity for macrophages and primary infection of tissue macrophages. The nature of sequence variation in the central region (vif, vpr, and vpx), the env gene, and the nef long terminal repeat (LTR) region in brain, colon, and other tissues was examined to see whether specific genetic changes were associated with SIV replication in brain or gut. Sequence analysis revealed strong conservation of the intergenic central region, nef, and the LTR. However, analysis of env sequences in these two macaques and one other revealed significant, interesting patterns of sequence variation. (i) Changes in env that were found previously to contribute to the replicative ability of SIVmac for macrophages in culture were present in the tissues of these animals. (ii) The greatest variability was located in the regions between V1 and V2 and from "V3" through C3 in gp120, which are different in location from the variable regions observed previously in animals with strong antibody responses and long-term persistent infection. (iii) The predominant sequence change of D-->N at position 385 in C3 is most surprising, since this change in both SIV and human immunodeficiency virus type 1 has been associated with dramatically diminished affinity for CD4 and replication in vitro. (iv) The nature of sequence changes at some positions (146, 178, 345, 385, and "V3") suggests that viral replication in brain and gut may be facilitated by specific sequence changes in env in addition to those that impart a general ability to replicate well in macrophages. These results demonstrate that complex selective pressures, including immune responses and varying cell and tissue specificity, can influence the nature of sequence changes in env.     

Immunization with a live, attenuated simian immunodeficiency virus vaccine leads to restriction of viral diversity in Rhesus macaques not protected from pathogenic challenge

Rhesus macaques immunized with simian immunodeficiency virus SIVmac239Deltanef but not protected from SIVmac251 challenge were studied to determine the genetic and biological characteristics of the breakthrough viruses. Assessment of SIV genetic diversity (env V1-V2) revealed a reduction in the number of viral species in the immunized, unprotected macaques, compared to the number in nonimmunized controls. However, no evidence for selection of a specific V1-V2 genotype was observed, and biologically cloned isolates from the animals with breakthrough virus were similar with respect to replication kinetics and coreceptor use in vitro.     

Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.     

Complex determinants of macrophage tropism in env of simian immunodeficiency virus.

Macrophage-tropic virus variants evolved during the course of infection of individual rhesus monkeys with cloned, non-macrophagetropic simian immunodeficiency virus. Specific changes in the envelope gene (env) were found to be primarily responsible for the dramatic increase in the ability of the virus to replicate in macrophages. Cloned viruses differing at nine amino acid positions in env exhibited a more than 100-fold difference in replicative capacity for primary cultures of rhesus monkey alveolar macrophages. At least five of the nine amino acid changes contributed to macrophage tropism. These determinants were distributed across the full length of env, including both the gp120 and gp41 products of the env gene. Furthermore, the emergence of macrophagetropic variants in vivo was associated with specific pathologic manifestations in which the macrophage is the major infected cell type. Thus, major determinants of macrophage tropism reside in env, they can be complex in nature, and the presence of macrophage-tropic virus variants in vivo can influence the disease course and disease manifestations.     

Rapid progression to simian AIDS can be accompanied by selection of CD4-independent gp120 variants with impaired ability to bind CD4

Aspartate 368 on human immunodeficiency virus type 1 (HIV-1) gp120 forms multiple contacts with CD4; in mutagenesis studies, its replacement by asparagine and corresponding changes in simian immunodeficiency virus SIVmac (D385N) reduced binding with CD4. Nevertheless, simian immunodeficiency virus envelopes with D385N were prevalent in several studies. Extending these observations, we also found D385N to be dominant among env clones from two rhesus macaques that progressed rapidly to simian AIDS. These envelopes showed a CD4-independent phenotype as well as reduced affinity to CD4. Moreover, an adjacent change, G383R, which was frequently coselected with D385N, further decreased binding. An optical biosensor study demonstrated that the SIVmac239 gp120 bound to CD4 with kinetics similar to those of HIV-1. However, the gp120s with D385N and G383R showed a 40-fold reduction in affinity, with a drastic increase in dissociation rate, indicating an inherently unstable complex. This finding showed that rapid progression to simian AIDS may be accompanied by the selection of CD4-independent gp120 variants with impaired CD4 binding ability.     

Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact.     

Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys.

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in amino acid changes. Variation in SU was not random but occurred predominantly in five discrete regions. Within these variable regions, a remarkable 98% of the nucleotide substitutions changed the amino acid. These results demonstrate that extensive sequence variability accumulates in vivo after infection with molecularly cloned virus and that selection occurs in vivo for changes in distinct variable regions in env.     

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.     

A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys.

The utility of the simian immunodeficiency virus of macaques (SIVmac) model of AIDS has been limited by the genetic divergence of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and the SIVs. To develop a better AIDS animal model, we have been exploring the infection of rhesus monkeys with chimeric simian/human immunodeficiency viruses (SHIVs) composed of SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rev. SHIV-89.6, constructed with the HIV-1 env of a cytopathic, macrophage-tropic clone of a patient isolate of HIV-1 (89.6), was previously shown to replicate to a high degree in monkeys during primary infection. However, pathogenic consequences of chronic infection were not evident. We now show that after two serial in vivo passages by intravenous blood inoculation of naive rhesus monkeys, this SHIV (SHIV-89.6P) induced CD4 lymphopenia and an AIDS-like disease with wasting and opportunistic infections. Genetic and serologic evaluation indicated that the reisolated SHIV-89.6P expressed envelope glycoproteins that resembled those of HIV-1. When inoculated into naive rhesus monkeys, SHIV-89.6P caused persistent infection and CD4 lymphopenia. This chimeric virus expressing patient isolate HIV-1 envelope glycoproteins will be valuable as a challenge virus for evaluating HIV-1 envelope-based vaccines and for exploring the genetic determinants of HIV-1 pathogenicity.