Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors.

UNLABELLED: African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo, while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4(+) T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4(+) T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals. IMPORTANCE: African green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptors in vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entry in vitro and may serve as entry coreceptors for SIVagm in vivo, since their mRNAs were detected in AGM memory CD4(+) T cells, the preferred target cells of SIV.

Characterization of simian immunodeficiency virus (SIV) that induces SIV encephalitis in rhesus macaques with high frequency: role of TRIM5 and major histocompatibility complex genotypes and early entry to the brain.

UNLABELLED: Although nonhuman primate models of neuro-AIDS have made tremendous contributions to our understanding of disease progression in the central nervous system (CNS) of human immunodeficiency virus type 1 (HIV-1)-infected individuals, each model holds advantages and limitations. In this study, in vivo passage of SIVsmE543 was conducted to obtain a viral isolate that can induce neuropathology in rhesus macaques. After a series of four in vivo passages in rhesus macaques, we have successfully isolated SIVsm804E. SIVsm804E shows efficient replication in peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) in vitro and induces neuro-AIDS in high frequencies in vivo. Analysis of the acute phase of infection revealed that SIVsm804E establishes infection in the CNS during the early phase of the infection, which was not observed in the animals infected with the parental SIVsmE543-3. Comprehensive analysis of disease progression in the animals used in the study suggested that host major histocompatibility complex class I (MHC-I) and TRIM5α genotypes influence the disease progression in the CNS. Taken together, our findings show that we have successfully isolated a new strain of simian immunodeficiency virus (SIV) that is capable of establishing infection in the CNS at early stage of infection and causes neuropathology in infected rhesus macaques at a high frequency (83%) using a single inoculum, when animals with restrictive MHC-I or TRIM5α genotypes are excluded. SIVsm804E has the potential to augment some of the limitations of existing nonhuman primate neuro-AIDS models. IMPORTANCE: Human immunodeficiency virus (HIV) is associated with a high frequency of neurologic complications due to infection of the central nervous system (CNS). Although the use of antiviral treatment has reduced the incidence of severe complications, milder disease of the CNS continues to be a significant problem. Animal models to study development of neurologic disease are needed. This article describes the development of a novel virus isolate that induces neurologic disease in a high proportion of rhesus macaques infected without the need for prior immunomodulation as is required for some other models.

TRIM5 alpha drives SIVsmm evolution in rhesus macaques.

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5(Q) was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5(TFP/TFP) or TRIM5(Cyp/TFP). Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5(TFP) restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5(Cyp). Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIM(TFP) alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.

Laser capture microdissection assessment of virus compartmentalization in the central nervous systems of macaques infected with neurovirulent simian immunodeficiency virus.

Nonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments.

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques.

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.

Development of neurological disease is associated with increased immune activation in simian immunodeficiency virus-infected macaques.

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.

Improved survival in rhesus macaques immunized with modified vaccinia virus Ankara recombinants expressing simian immunodeficiency virus envelope correlates with reduction in memory CD4+ T-cell loss and higher titers of neutralizing antibody.

Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4(+) T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4(+) T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4(+) T-cell loss.

Correction of a carboxyl terminal simian immunodeficiency virus Nef frameshift mutation restores virus replication in macaques.

Previous studies demonstrated that the nef gene is a critical determinant of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. In the present study, we evaluated the effect of a spontaneous frameshift mutation in the C-terminus of the nef gene of the minimally pathogenic SIVsmH4i clone. This clone exhibited a single nucleotide deletion in the nef gene relative to pathogenic SIV clones that resulted in a frameshift and addition of 46 amino acids to the C-terminus of Nef. We generated a corrected version of this clone, SIVsmH4i Nef+ that restored Nef protein expression. Inoculation of macaques with SIVsmH4i resulted in delayed and low levels of peak viremia. This contrasted with improved kinetics and robust peak viremia in macaques inoculated with the corrected version. Despite the restoration of in vivo replication ability, neither clone resulted in memory CD4+ T cell loss or disease in a period of two years.

Adaptive evolution of simian immunodeficiency viruses isolated from 2 conventional-progressor macaques with encephalitis.

Simian immunodeficiency virus-infected macaques may develop encephalitis, a feature more commonly observed in macaques with rapid progressive disease than in those with conventional disease. In this report, an analysis of 2 conventional progressors with encephalitis is described. Phylogenetic analyses of viruses isolated from the cerebrospinal fluid and plasma of both macaques demonstrated compartmentalization. Furthermore, these viruses appear to have undergone adaptive evolution to preferentially replicate in their respective cell targets of monocyte-derived macrophages and peripheral blood mononuclear cells. A statistically significant loss of potential N-linked glycosylation sites in glycoprotein 160 was observed in viruses isolated from the central nervous system.

A rapid progressor-specific variant clone of simian immunodeficiency virus replicates efficiently in vivo only in the absence of immune responses.

A subset of simian immunodeficiency virus (SIV)-infected macaques progresses rapidly to disease with transient SIV-specific immune responses and high viral loads. Unique SIV variants with convergent Env mutations evolve in these rapid progressor (RP) macaques. To address the pathogenic significance of RP-specific variants, we generated infectious molecular clones from the terminal-phase plasma of an RP macaque. Inoculation of macaques with a representative clone, SIVsmH635FC, resulted in a persistent viremia, comparable to that produced by pathogenic SIVsmE543-3, and a chronic disease with progressive loss of CD4(+) T cells. However, SIVsmH635FC did not reproduce the rapid-disease phenomenon. Molecular analyses of viruses from these macaques revealed rapid reversion to the wild-type SIVsmE543-3 sequence at two RP-specific sites and slower reversion at another three sites. SIVsmH635FC infection was not sufficient to cause rapid progression even following coinoculation with SIVsmE543-3, despite acute depletion of memory CD4(+) T cells. SIVsmH635FC competed efficiently during primary infection in the coinoculated macaques, but SIVsmE543-3 predominated after the development of SIV-specific immune responses. These data suggest that the replication fitness of the RP variant was similar to that of SIVsmE543-3 in a naïve host; however, SIVsmH635FC was at a disadvantage following the development of SIV-specific immune responses. Consistent with these findings, neutralization assays revealed that SIVsmH635FC was highly sensitive to neutralization but that the parental SIVsmE543-3 strain was highly resistant. This study suggests that the evolution of RP-specific variants is the result of replication in a severely immunocompromised host, rather than the direct cause of rapid progression.

Loss of naïve cells accompanies memory CD4+ T-cell depletion during long-term progression to AIDS in Simian immunodeficiency virus-infected macaques.

Human immunodeficiency virus and simian immunodeficiency virus (SIV) induce a slow progressive disease, characterized by the massive loss of memory CD4+ T cells during the acute infection followed by a recovery phase in which virus replication is partially controlled. However, because the initial injury is so severe and virus production persists, the immune system eventually collapses and a symptomatic fatal disease invariably occurs. We have assessed CD4+ T-cell dynamics and disease progression in 12 SIV-infected rhesus monkeys for nearly 2 years. Three macaques exhibiting a rapid progressor phenotype experienced rapid and irreversible loss of memory, but not naïve, CD4+ T lymphocytes from peripheral blood and secondary lymphoid tissues and died within the first 6 months of virus inoculation. In contrast, SIV-infected conventional progressor animals sustained marked but incomplete depletions of memory CD4+ T cells and continuous activation/proliferation of this T-lymphocyte subset. This was associated with a profound loss of naïve CD4+ T cells from peripheral blood and secondary lymphoid tissues, which declined at rates that correlated with disease progression. These data suggest that the persistent loss of memory CD4(+)T cells, which are being eliminated by direct virus killing and activation-induced cell death, requires the continuous differentiation of naïve into memory CD4+ T cells. This unrelenting replenishment process eventually leads to the exhaustion of the naïve CD4+T-cell pool and the development of disease.

Immunodeficiency in the absence of high viral load in pig-tailed macaques infected with Simian immunodeficiency virus SIVsun or SIVlhoest.

Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4(+) lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as Mycobacterium avium or Pneumocystis carinii infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.

Characterization and comparison of recombinant simian immunodeficiency virus from drill (Mandrillus leucophaeus) and mandrill (Mandrillus sphinx) isolates.

Since simian immunodeficiency virus (SIV) was found to be the source of the human AIDS pandemic, a major goal has been to characterize the diversity of SIV strains in the wild and to assess their potential for crossover into humans. In the present study, SIV was isolated from a seropositive drill (Mandrillus leucophaeus) and three seropositive mandrills (Mandrillus sphinx) by using macaque peripheral blood mononuclear cells (PBMC). Full-length sequences were obtained from a drill and mandrill and designated SIVdrl1FAO and SIVmnd5440, respectively. A 182-bp fragment of the pol genes of the two remaining mandrill SIV isolates was also analyzed. Phylogenetic analyses demonstrated that SIVdrl1FAO formed a monophyletic clade with SIVmnd5440 and SIVmndM14, recently designated SIVmnd type 2. Both the SIVdrl and SIVmnd type 2 genomes carried a vpx gene and appeared to share a common ancestor with SIVrcm in the 5' region of the genome and with SIVmndGB1 (type 1) in the 3' region of the genome. A statistically significant recombination breakpoint was detected at the beginning of envelope, suggesting that the viruses were descendents of the same recombinant. Phylogenetic analysis of vpx and vpr genes demonstrated that the vpx genes formed a monophyletic cluster that grouped with vpr from SIVagm. In addition, both SIVdrl1FAO and SIVmnd5440 replicated in human PBMC and therefore could pose a risk of transmission to the human population.

Highly pathogenic SHIVs and SIVs target different CD4+ T cell subsets in rhesus monkeys, explaining their divergent clinical courses.

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 3-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause a complete, irreversible, and systemic depletion of CD4(+) T lymphocytes in rhesus monkeys within weeks of infection. By using small-molecule competitors specific for CCR5 and CXCR4 in ex vivo assays, we found that highly pathogenic SHIV(DH12R) exclusively uses CXCR4 for infection of rhesus peripheral blood mononuclear cells, whereas SIV(mac239) and SIV(smE543) use CCR5 for entry into the same cells. During the period of peak virus production in SHIV(DH12R)- or SHIV(89.6P)-infected rhesus monkeys, massive elimination of CXCR4(+) naïve CD4(+) T cells occurred. In contrast, circulating CCR5(+) memory CD4(+) T cells were selectively depleted in rapidly progressing SIV-infected monkeys. At the time of their death, two SIV rapid progressors had experienced a nearly complete loss of the memory CD4(+) T cell subset from the blood and mesenteric lymph nodes. Thus, pathogenic SHIVs and SIVs target different subsets of CD4(+) T cells in vivo, with the pattern of CD4(+) T lymphocyte depletion being inextricably linked to chemokine receptor use. In the context of developing an effective prophylactic vaccine, which must potently control virus replication during the primary infection, regimens that suppress SHIVs might not protect monkeys against SIV or humans against HIV-1.