Welkin E. Johnson

Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIVmac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIVsmm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.

TRIM5 Suppresses Cross-Species Transmission of a Primate Immunodeficiency Virus and Selects for Emergence of Resistant Variants in the New Species

Cross-species transmission of simian immunodeficiency virus from sooty mangabeys (SIVsm) into rhesus macaques, and subsequent emergence of pathogenic SIVmac, required adaptation to overcome restriction encoded by the macaque TRIM5 gene.

Dissecting the Neutralizing Antibody Specificities of Broadly Neutralizing Sera from Human Immunodeficiency Virus Type 1-Infected Donors

ABSTRACT Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.

Evolution of a TRIM5-CypA splice isoform in old world monkeys

The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5α has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3′ splice site upstream of exon 7, which may prevent or reduce expression of the α-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares ∼80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5–10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5α

Retroviral restriction factor TRIM5α exhibits a high degree of sequence variation among primate species. It has been proposed that this diversity is the cumulative result of ancient, lineage-specific episodes of positive selection. Here, we describe the contribution of within-species variation to the evolution of TRIM5α. Sampling within two geographically distinct Old World monkey species revealed extensive polymorphism, including individual polymorphisms that predate speciation (shared polymorphism). In some instances, alleles were more closely related to orthologues of other species than to one another. Both silent and nonsynonymous changes clustered in two domains. Functional assays revealed consequences of polymorphism, including differential restriction of a small panel of retroviruses by very similar alleles. Together, these features indicate that the primate TRIM5α locus has evolved under balancing selection. Except for the MHC there are few, if any, examples of long-term balancing selection in primates. Our results suggest a complex evolutionary scenario, in which fixation of lineage-specific adaptations is superimposed on a subset of critical polymorphisms that predate speciation events and have been maintained by balancing selection for millions of years.

Assorted Mutations in the Envelope Gene of Simian Immunodeficiency Virus Lead to Loss of Neutralization Resistance against Antibodies Representing a Broad Spectrum of Specificities

ABSTRACT Simian immunodeficiency virus (SIV) of macaques isolate SIVmac239 is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, a property that it shares with most primary isolates of human immunodeficiency virus type 1 (HIV-1). This resistance is important for the ability of the virus to persist at high levels in vivo. To explore the physical features of the viral envelope complex that contribute to the neutralization-resistant phenotype, we examined a panel of SIVmac239 derivatives for sensitivity to neutralization by a large collection of monoclonal antibodies (MAbs). These MAbs recognize both linear and conformational epitopes throughout the viral envelope proteins. The variant viruses included three derivatives of SIVmac239 with substitutions in specific N-linked glycosylation sites of gp120 and a fourth variant that lacked the100 amino acids that encompass the V1 and V2 loops. Also included in this study was SIVmac316, a variant of SIVmac239 with distributed mutations in env that confer significantly increased replicative capacity in tissue macrophages. These viruses were chosen to represent a broad range of neutralization sensitivities based on susceptibility to pooled, SIV-positive plasma. All three of these very different kinds of mutations (amino acid substitutions, elimination of N-glycan attachment sites, and a 100-amino-acid deletion spanning variable loops V1 and V2) dramatically increased sensitivity to neutralization by MAbs from multiple competition groups. Thus, the mutations did not simply expose localized epitopes but rather conferred global increases in neutralization sensitivity. The removal of specific N-glycan attachment sites from V1 and V2 led to increased sensitivity to neutralization by antibodies recognizing epitopes from both within and outside of the V1-V2 sequence. Surprisingly, while most of the mutations that gave rise to increased sensitivity were located in the N-terminal half of gp120 (surface subunit [SU]), the greatest increases in sensitivity were to MAbs recognizing the C-terminal half of gp120 or the ectodomain of gp41 (transmembrane subunit [TM]). This reagent set and information should now be useful for defining the physical, structural, thermodynamic, and kinetic factors that influence relative sensitivity to antibody-mediated neutralization.

Analysis of copy number variation in the rhesus macaque genome identifies candidate loci for evolutionary and human disease studies

Copy number variants (CNVs) are heritable gains and losses of genomic DNA in normal individuals. While copy number variation is widely studied in humans, our knowledge of CNVs in other mammalian species is more limited. We have designed a custom array-based comparative genomic hybridization (aCGH) platform with 385 000 oligonucleotide probes based on the reference genome sequence of the rhesus macaque (Macaca mulatta), the most widely studied non-human primate in biomedical research. We used this platform to identify 123 CNVs among 10 unrelated macaque individuals, with 24% of the CNVs observed in multiple individuals. We found that segmental duplications were significantly enriched at macaque CNV loci. We also observed significant overlap between rhesus macaque and human CNVs, suggesting that certain genomic regions are prone to recurrent CNV formation and instability, even across a total of approximately 50 million years of primate evolution ( approximately 25 million years in each lineage). Furthermore, for eight of the CNVs that were observed in both humans and macaques, previous human studies have reported a relationship between copy number and gene expression or disease susceptibility. Therefore, the rhesus macaque offers an intriguing, non-human primate outbred model organism with which hypotheses concerning the specific functions of phenotypically relevant human CNVs can be tested.

Prevention of Infection by a Granulocyte-Macrophage Colony-Stimulating Factor Co-Expressing DNA/Modified Vaccinia Ankara Simian Immunodeficiency Virus Vaccine

A simian immunodeficiency virus (SIV) vaccine coexpressing granulocyte-macrophage colony stimulating factor (GM-CSF) prevented infection in 71% of macaques that received 12 rectal challenges. The SIVsmE660 challenge had the tropism of incident human immunodeficiency virus (HIV) infections and a similar genetic distance from the SIV239 vaccine as intraclade HIV isolates. The heterologous prime-boost vaccine regimen used recombinant DNA for priming and recombinant modified vaccinia Ankara for boosting. Co-expression of GM-CSF in the DNA prime enhanced the avidity of elicited immunoglobulin G for SIV envelope glycoproteins, the titers of neutralizing antibody for easy-to-neutralize SIV isolates, and antibody-dependent cellular cytotoxicity. Impressively, the co-expressed GM-CSF increased vaccine-induced prevention of infection from 25% in the non-GM-CSF co-expressing vaccine group to 71% in the GM-CSF co-expressing vaccine group. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r = 0.9; P < .0001).

Molecular evolution of the antiretroviral TRIM5 gene

In 2004, the first report of TRIM5α as a cellular antiretroviral factor triggered intense interest among virologists, particularly because some primate orthologs of TRIM5α have activity against HIV. Since that time, a complex and eventful evolutionary history of the TRIM5 locus has emerged. A review of the TRIM5 literature constitutes a veritable compendium of evolutionary phenomena, including elevated rates of nonsynonymous substitution, divergence in subdomains due to short insertions and deletions, expansions and contractions in gene copy number, pseudogenization, balanced polymorphism, trans-species polymorphism, convergent evolution, and the acquisition of new domains by exon capture. Unlike most genes, whose history is dominated by long periods of purifying selection interspersed with rare instances of genetic innovation, analysis of restriction factor loci is likely to be complicated by the unpredictable and more-or-less constant influence of positive selection. In this regard, the molecular evolution and population genetics of restriction factor loci most closely resemble patterns that have been documented for immunity genes, such as class I and II MHC genes, whose products interact directly with microbial targets. While the antiretroviral activity encoded by TRIM5 provides plausible mechanistic hypotheses for these unusual evolutionary observations, evolutionary analyses have reciprocated by providing significant insights into the structure and function of the TRIM5α protein. Many of the lessons learned from TRIM5 should be applicable to the study of other restriction factor loci, and molecular evolutionary analysis could facilitate the discovery of new antiviral factors, particularly among the many TRIM genes whose functions remain as yet unidentified.