Phylogenetic study of SIVcpz MT145 virus based on proteome and genome analysis

Molecular phylogenetic studies were performed by the alignment of protein/nucleotide sequences of human/simian immunodeficiency virus (HIV/SIV), followed by the construction of phylograms according to maximum likelihood method. We aimed to investigate the evolutionary relationship of the recombinant SIVcpzMT145, to other well-known SIVcpz and HIV-1 viruses. Expectedly, MT145 follows the rule of feasible recombination occurrence in SIVcpz clade as to it consists several recombinations in different genome sites including gag, Pol, and Env region. Phylograms indicated that in Pol gene, MT145 is more related to GAB1 and CAM13 SIVs; while in Env gene, it has a closed relationship to GAB2 SIV. Moreover, MT145 differs from other SIVcpzPtt strains in the Env V3 loop having the QIGPAMT motif (same as HIV-1N), instead of usual QIGPGMT motif in these strains. Data indicated that the Env proteins contain considerable amino acid sequence diversification. Overall, this study suggests that, parts of the Gag and especially Vpu/Env gene sequences of SIVcpzMT145 were derived from an unknown SIVcpz lineage ancestral to HIV-1 group M/MB66.     

Characterization of a novel simian immunodeficiency virus with a vpu gene from greater spot-nosed monkeys (Cercopithecus nictitans) provides new insights into simian/human immunodeficiency virus phylogeny

In the present study, we describe a new simian immunodeficiency virus (SIV), designated SIVgsn, naturally infecting greater spot-nosed monkeys (Cercopithecus nictitans) in Cameroon. Together with SIVsyk, SIVgsn represents the second virus isolated from a monkey belonging to the Cercopithecus mitis group of the Cercopithecus genus. Full-length genome sequence analysis of two SIVgsn strains, SIVgsn-99CM71 and SIVgsn-99CM166, revealed that despite the close phylogenetic relationship of their hosts, SIVgsn was highly divergent from SIVsyk. First of all, they differ in their genomic organization. SIVgsn codes for a vpu homologue, so far a unique feature of the members of the SIVcpz/human immunodeficiency virus type 1 (HIV-1) lineage, and detailed phylogenetic analyses of various regions of the viral genome indicated that SIVgsn might be a mosaic of sequences with different evolutionary histories. SIVgsn was related to SIVsyk in Gag and part of Pol and related to SIVcpz in Env, and the middle part of the genome did not cluster significantly with any of the known SIV lineages. When comparing the two SIVgsn Env sequences with that of SIVcpz, a remarkable conservation was seen in the V3 loop, indicating a possible common origin for the envelopes of these two viruses. The habitats of the two subspecies of chimpanzees infected by SIVcpz overlap the geographic ranges of greater spot-nosed monkeys and other monkey species, allowing cross-species transmission and recombination between coinfecting viruses. The complex genomic structure of SIVgsn, the presence of a vpu gene, and its relatedness to SIVcpz in the envelope suggest a link between SIVgsn and SIVcpz and provide new insights about the origin of SIVcpz in chimpanzees.     

Characterization of a novel simian immunodeficiency virus (SIVmonNG1) genome sequence from a mona monkey (Cercopithecus mona)

A novel simian immunodeficiency virus (SIV) sequence has been recovered from RNA extracted from the serum of a mona monkey (Cercopithecus mona) wild born in Nigeria. The sequence was obtained by using novel generic (degenerate) PCR primers and spans from two-thirds into the gag gene to the 3' poly(A) tail of the SIVmonNG1 RNA genome. Analysis of the open reading frames revealed that the SIVmonNG1 genome codes for a Vpu protein, in addition to Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef proteins. Previously, only lentiviruses infecting humans (human immunodeficiency virus type 1 [HIV-1]) and chimpanzees (SIVcpz) were known to have a vpu gene; more recently, this has also been found in SIVgsn from Cercopithecus nictitans. Overall, SIVmonNG1 most closely resembles SIVgsn: the env gene sequence groups with HIV-1/SIVcpz env sequences, whereas the pol gene sequence clusters closely with the pol sequence of SIVsyk from Cercopithecus albogaris. By bootscanning and similarity plotting, the first half of pol resembles SIVsyk, whereas the latter part is closer to SIVcol from Colobus guereza. The similarities between the complex mosaic genomes of SIVmonNG1 and SIVgsn are consistent with a shared or common lineage. These data further highlight the intricate nature of the relationships between the SIVs from different primate species and will be helpful for unraveling these associations.     

Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.     

A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV_cpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV_cpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.     

Natural infection of chimpanzees with new lentiviruses related to HIV-1/SIVcpz

To determine newly identified lentiviruses, termed simian immunodeficiency virus (SIV)cpz97CG4 and SIVcpz97CG6, from two wild-captured juvenile brother chimpanzees in the Republic of Congo, subgenomic pol (integrase, 288 bp), 5'tat/rev-env Cl (including vpu, 354 bp) and env (C2-C4, 544 bp) gene fragments were amplified and sequenced. The analysis revealed significantly discordant phylogenetic positions of SIVcpz97CG in each genomic region. In the trees derived from partial env sequences (V3), both SIVcpz strains clustered in human immunodeficiency virus type 1 (HIV-1) subtype A. However, in the trees derived from partial pol (integrase) and 5'tat/rev-env C1 (including vpu) sequences, they clustered independently from any of the known HIV-1 subtypes. Especially, in the 5'tat/rev-vpu tree, they branched before the root of HIV-1 group M. These findings suggest that these Congolese SIVcpz genomes are mosaic, probably due to a recombinational event in the recent past, and it provides evidence for a rather recently occurring cross-species transmission between humans and chimpanzees.     

A Novel Simian Immunodeficiency Virus (SIVdrl) pol Sequence from the Drill Monkey, Mandrillus leucophaeus

The drill monkey has been shown by serology and PCR to harbor a unique simian immunodeficiency virus (SIVdrl). A pol sequence, amplified from uncultured peripheral blood cells, is most closely related to the equivalent SIV sequences from the red-capped mangabey (SIVrcm), the sabaeus African green monkey (SIVagmSAB), and the chimpanzee (SIVcpz) and to the human immunodeficiency virus type 1 (HIV-1) sequence of humans. It is as yet unclear whether SIVdrl has a mosaic genome like SIVrcm and SIVagmSAB, is a member of the SIVcpz/HIV-1 lineage, or represents a novel primate lentivirus lineage.     

Stable expression of primary human immunodeficiency virus type 1 structural gene products by use of a noncytopathic sindbis virus vector

Efficient expression of the human immunodeficiency virus type 1 (HIV-1) structural gene products Gag, Pol, and Env involves the regulation by viral Rev and Rev-responsive elements (RRE). Removal of multiple inhibitory sequences (INS) in the coding regions of these structural genes or modification of the codon usage patterns of HIV-1 genes to those used by highly expressed human genes has been found to significantly increase HIV-1 structural protein expression in the absence of Rev and RRE. In this study, we show that efficient and stable expression of the HIV-1 structural gene products Gag and Env could be achieved by transfection with a noncytopathic Sindbis virus expression vector by using HIV-1 sequences from primary isolates without any sequence modification. Stable expression of these Gag and Env proteins was observed for more than 12 months. The fact that the Sindbis virus expression vector replicates its RNA only in the cytoplasm of the transfected cells and the fact that the lack of expression of HIV-1 Gag by the DNA vector containing unmodified HIV-1 gag sequences was associated with a lack of detectable cytoplasmic gag RNA suggest that a major blockage in the expression of HIV-1 structural proteins in the absence of Rev/RRE is caused by inefficient accumulation of mRNA in the cytoplasm. Efficient long-term expression of structural proteins of diverse HIV-1 strains by the noncytopathic Sindbis virus expression system may be a useful tool for functional study of HIV-1 gene products and vaccine research.     

Analysis of the evolutionary relationships of HIV-1 and SIVcpz sequences using bayesian inference: implications for the origin of HIV-1

The most plausible origin of HIV-1 group M is an SIV lineage currently represented by SIVcpz isolated from the chimpanzee subspecies Pan troglodytes troglodytes. The origin of HIV-1 group O is less clear. Putative recombination between any of the HIV-1 and SIVcpz sequences was tested using bootscanning and Bayesian-scanning plots, as well as a new method using a Bayesian multiple change-point (BMCP) model to infer parental sequences and crossing-over points. We found that in the case of highly divergent sequences, such as HIV-1/SIVcpz, Bayesian scanning and BMCP methods are more appropriate than bootscanning analysis to investigate spatial phylogenetic variation, including estimating the boundaries of the regions with discordant evolutionary relationships and the levels of support of the phylogenetic clusters under study. According to the Bayesian scanning plots and BMCP method, there was strong evidence for discordant phylogenetic clustering throughout the genome: (1) HIV-1 group O clustered with SIVcpzANT/TAN in middle pol, and partial vif/env; (2) SIVcpzGab1 clustered with SIVcpzANT/TAN in 3'pol/vif, and middle env; (3) HIV-1 group O grouped with SIVcpzCamUS and SIVcpzGab1 in p17/p24; (4) HIV-1 group M was more closely related to SIVcpzCamUS in 3'gag/pol and in middle pol, whereas in partial gp120 group M clustered with group O. Conditionally independent phylogenetic analysis inferred by maximum likelihood (ML) and Bayesian methods further confirmed these findings. The discordant phylogenetic relationships between the HIV-1/SIVcpz sequences may have been caused by ancient recombination events, but they are also due, at least in part, to altered rates of evolution between parental SIVcpz lineages.     

Co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in HIV-1 ancestor

Cross-species transmission and adaptation of simian immunodeficiency viruses (SIVs) to humans have given rise to human immunodeficiency viruses (HIVs). HIV type 1 (HIV-1) and type 2 (HIV-2) were derived from SIVs that infected chimpanzee (SIVcpz) and sooty mangabey (SIVsm), respectively. The HIV-1 restriction factor SAMHD1 inhibits HIV-1 infection in human myeloid cells and can be counteracted by the Vpx protein of HIV-2 and the SIVsm lineage. However, HIV-1 and its ancestor SIVcpz do not encode a Vpx protein and HIV-1 has not evolved a mechanism to overcome SAMHD1-mediated restriction. Here we show that the co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in SIVcpz and HIV-1. We found evidence for positive selection of SAMHD1 in orangutan, gibbon, rhesus macaque, and marmoset, but not in human, chimpanzee and gorilla that are natural hosts of Vpx-negative HIV-1, SIVcpz and SIVgor, respectively, indicating that vpx drives the evolution of primate SAMHD1. Ancestral host state reconstruction and temporal dynamic analyses suggest that the most recent common ancestor of SIVrcm, SIVmnd, SIVcpz, SIVgor and HIV-1 was a SIV that had a vpx gene; however, the vpx gene of SIVcpz was lost approximately 3643 to 2969 years ago during the infection of chimpanzees. Thus, HIV-1 could not inherit the lost vpx gene from its ancestor SIVcpz. The lack of Vpx in HIV-1 results in restricted infection in myeloid cells that are important for antiviral immunity, which could contribute to the AIDS pandemic by escaping the immune responses.     

Characterization of a novel vpu-harboring simian immunodeficiency virus from a Dent's Mona monkey (Cercopithecus mona denti)

We report the identification of a new simian immunodeficiency virus (SIV), designated SIVden, in a naturally infected Dent's Mona monkey (Cercopithecus mona denti), which was kept as pet in Kinshasa, capital of the Democratic Republic of Congo. SIVden is genetically distinct from the previously characterized primate lentiviruses. Analysis of the full-length genomic sequence revealed the presence of a vpu open reading frame. This gene is also found in the virus lineage of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus (SIVcpz) and was recently described in viruses isolated from Cercopithecus nictitans, Cercopithecus mona, and Cercopithecus cephus. The SIVden vpu coding region is shorter than the HIV-1/SIVcpz and the SIVgsn, SIVmon, and SIVmus counterparts. Unlike Pan troglodytes schweinfurthii viruses (SIVcpzPts) and Cercopithecus monkey viruses (SIVgsn, SIVmon, and SIVmus), the SIVden Vpu contains the characteristic DSGXES motif which was shown to be involved in Vpu-mediated CD4 and IkappaBalpha proteolysis in HIV-1 infected cells. Although it harbors a vpu gene, SIVden is phylogenetically closer to SIVdeb isolated from De Brazza's monkeys (Cercopithecus neglectus), which lacks a vpu gene, than to Cercopithecus monkey viruses, which harbor a vpu sequence.     

The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin adaptor [correction of adapter

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.     

SIVcpz cross-species transmission and viral evolution toward HIV-1 in a humanized mouse model

HIV-1 evolved from its progenitor SIV strains, but details are lacking on its adaptation to the human host. We followed the evolution of SIVcpz in humanized mice to mimic cross-species transmission. Increasing viral loads, CD4⁺ T-cell decline, and non-synonymous mutations were seen in the entire genome reflecting viral adaptation.     

Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.     

Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.     

Distinguishing features of an infectious molecular clone of the highly divergent and noncytopathic human immunodeficiency virus type 2 UC1 strain.

A full-length infectious molecular clone was derived from the noncytopathic human immunodeficiency virus type 2 UC1 strain (HIV-2UC1) that was originally recoverd from an individual from the Ivory Coast. Like the parental isolate, the molecularly cloned virus (HIV-2UC1mc or UC1 mc) demonstrates a reduced ability to induce syncytium formation, to kill cells, and to down-modulate the cell surface CD4 receptor in infected cells. Phylogenetic analysis of the DNA sequence of UC1mc revealed that it is the first full-length infectious molecular clone in the second HIV-2 subgroup previously identified by partial sequence analysis of the HIV-2D205 and HIV-2GH-2 strains. These highly divergent HIV-2 strains appear to be genetically equidistant from other HIV-2 and simian immunodeficiency virus SIVmac/sm strains. UC1mc is unlike any other HIV-2 or SIVmac/sm strain in that it lacks a cysteine residue at the proposed signal peptide cleavage site in Env. However, site-directed mutagenesis experiments indicate that this missing cysteine is not alone important in the noncytopathic phenotype of UC1mc. Like other HIV-2 and SIV strains, the UC1mc Env transmembrane protein (gp43) is mutated to a truncated form (gp34) after passage in certain T-cell lines. The UC1 molecular clone should be helpful in determining the genetic sequences associated with HIV-2 cytopathicity.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.     

The simian immunodeficiency virus envelope glycoprotein contains multiple signals that regulate its cell surface expression and endocytosis

The cell surface expression of the envelope glycoproteins (Envs) of primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located in the Env cytoplasmic domain. Here, we show that a membrane proximal signal that directs the simian immunodeficiency virus (SIV) Env to clathrin‐coated pits, and is conserved in all SIV and human immunodeficiency virus Envs, conforms to a Yxxø motif (where x can be any amino acid and Ø represents a large hydrophobic residue). This motif is similar to that described for a number of cellular membrane proteins. By surface plasmon resonance we detected a high affinity interaction between peptides containing this membrane proximal signal and both AP1 and AP2 clathrin adaptor complexes. Mutation of the tyrosine in this membrane proximal motif in a SIV Env with a prematurely truncated cytoplasmic domain leads to a ≥25‐fold increase in Env expression on infected cells. By contrast, the same mutation in an Env with a full‐length cytoplasmic domain increases cell surface expression only 4‐fold. We show that this effect results from the presence of additional endocytosis signals in the full‐length cytoplasmic domain. Chimeras containing CD4 ecto‐ and membrane spanning domains and a full‐length SIV Env cytoplasmic domain showed rapid endocytosis even when the membrane proximal tyrosine‐based signal was disrupted. Mapping experiments indicated that at least some of the additional endocytosis information is located between residues 743 and 812 of Env from the SIV_mac239 molecular clone. Together, our findings indicate that the cytoplasmic domain of SIV Env contains multiple endocytosis and/or trafficking signals that modulate its surface expression on infected cells, and suggest an important role for this function in pathogenesis.     

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.