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Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+T-lymphocyte loss.

Five potent and broadly anti-HIV neutralizing monoclonal antibodies are able to block infection by two different SHIVs in monkeys. The authors show that antibodies targeting the outer glycan coat were the most effective and determined that titers of roughly 1:100 protected half the animals., It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti–HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239gag-poland HIV-1 89.6envelicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV DH12 ) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups ( P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV DH12 strongly correlated with the level of NAbs directed against the virus on the day of challenge ( P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV DH12 challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) ( P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV DH12R induces a systemic depletion of CD4 + T lymphocytes in rhesus monkeys during the initial 3–4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2–5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4 + T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV DH12R infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo . They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

Virus–specific antibodies protect individuals against a wide variety of viral infections1–7. To assess whether human immunodeficiency virus type 1 (HIV–1) envelope–specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV–1 isolates, and used this for passive immunization of pig–tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian–human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV–1DH12, a dual–tropic primary virus isolate. Here we show that anti–SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody–mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV–challenged macaques was in the range of 1:5–1:8. The HIV–1–specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non–neutralizing anti–HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Alleles at the Fv1 gene of inbred mice confer resistance to infection and spread of vertically or horizontally transmitted murine leukemia viruses (MuLV). The nucleotide sequence of Fv1 bears similarity to the gag of a human endogenous retrovirus, HERV-L, but is more closely related to the gag-coding sequence of a newly described class of HERV-L-related mouse endogenous retroviruses designated MuERV-L. Both observations suggest an origin of Fv1 from endogenous gag sequences. The molecular definition of Fv1 provided an opportunity to determine the phylogeny of the gene among wild mice and its relation to MuERV-L. PCR primers, chosen to include most of the coding region of Fv1 for both the n and b alleles, were used to amplify sequences from animals of the genus Mus, which were then sequenced. Closely related products were obtained from almost all animals examined that evolved after the separation from Rattus, in which the homologous gene was shown to be absent. A phylogenetic tree generated with Fv1 sequence data differs noticeably from that developed with sequence data from other genes. In addition, non-synonymous changes were found to be present twice as frequently as synonymous changes, a fact that departs from the standard behavior of a structural gene. These observations suggest that the Fv1 gene may have been subjected to possible horizontal transfers as well as to positive Darwinian selection.

Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3ϵ marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1β (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-γ (476 bp), and TNF-α (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3ϵ), immune response induction (IL-1β, IL-2), TH subsets (TH1, IL-2/IFN-γ vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-γ, TNF-α). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-γ and TNF-α RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection. J. Med. Virol. 53:85–95, 1997. © 1997 Wiley-Liss, Inc.

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9-14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5 weeks in some long-term chronically SHIV-infected animals with low CD4(+) T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV AD8 ) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.

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 TRIM5Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5TFP/TFP or TRIM5Cyp/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 TRIM5TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5Cyp. 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 TRIMTFP 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., Author Summary Human immunodeficiency virus (HIV) resulted from the transmission of simian immunodeficiency viruses (SIV) from nonhuman primates followed by adaptation and expansion as a pandemic in humans. This required the virus to overcome a variety of intrinsic host restriction factors in humans in order to replicate efficiently. Similarly, SIV encounters restriction factors upon cross-species transmission between nonhuman primates, specifically from a natural host species such as sooty mangabey monkeys to rhesus macaques. Previously we observed significant differences in the levels of virus replication of SIV among rhesus macaques due to subtle differences in one of these restriction factors, TRIM5 among individual macaques. Although a restrictive version of TRIM5 resulted in lower viremia, we also observed that the virus spontaneously mutated in the viral capsid gene and that these mutations were associated with escape from TRIM5 restriction. In the present study, we found that introduction of these escape mutations into the parental virus confers resistance to TRIM5 both in tissue culture and in macaques. These studies provide direct evidence that TRIM5 is a critical factor influencing the cross-species transmission of SIV in primates.

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIV AD8-EO ) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1 DH12 strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIV AD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIV AD8 -infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti–HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41–51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIV AD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains.

Fresh human immunodeficiency virus type 1 (HIV-1) isolates from patients with AIDS were screened for infectivity in chimpanzee peripheral blood mononuclear cells (PBMC) to identify strains potentially able to generate high virus loads in an inoculated animal. Only 3 of 23 isolates obtained were infectious in chimpanzee cells. Of these three, only one (HIV-1DH12) was able to initiate a productive infection in PBMC samples from all 25 chimpanzees tested. HIV-1DH12 tissue culture infections were characterized by extremely rapid replication kinetics, profound cytopathicity, and tropism for chimp and human PBMC, primary human macrophage, and several human T-cell lines. An infection was established within 1 week of inoculating a chimpanzee with 50 50% tissue culture infective doses of HIV-1DH12; cell-free virus was recovered from the plasma at weeks 1, 2, and 4 and was associated with the development of lymphadenopathy. Virus loads during the primary infection and at 6 months postinoculation were comparable to those reported in HIV-1-seropositive individuals.

The matrix protein of human immunodeficiency virus type 1 is encoded by the amino-terminal portion of the Gag precursor and is postulated to be involved in a variety of functions in the virus life cycle. To define domains and specific amino acid residues of the matrix protein that are involved in virus particle assembly, we introduced 35 amino acid substitution mutations in the human immunodeficiency virus type 1 matrix protein. Using reverse transcriptase and radioimmunoprecipitation analyses and transmission electron microscopy, we assessed the mutants for their ability to form virus particles and to function in the infection process. This study has identified several domains of the matrix protein in which single amino acid substitutions dramatically reduce the efficiency of virus particle production. These domains include the six amino-terminal residues of matrix, the region of matrix between amino acids 55 and 59, and the region between amino acids 84 and 95. Single amino acid substitutions in one of these domains (between matrix amino acids 84 and 88) result in a redirection of the majority of virus particle formation to sites within cytoplasmic vacuoles.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity., link_to_OA_fulltext

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.

The progressive decline of CD4+ T cells is a hallmark of disease progression in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection. Whereas the acute phase of the infection is dominated by virus-mediated depletion of memory CD4+ T cells, chronic infection is often associated with a progressive decline of total CD4+ T cells, including the naïve subset. The mechanism of this second phase of CD4+ T cell loss is unclear and may include immune activation–induced cell death, immune-mediated destruction, and regenerative or homeostatic failure. We studied patterns of CD4+ T cell subset depletion in blood and tissues in a group of 20 rhesus macaques inoculated with derivatives of the pathogenic SIVsmE543-3 or SIVmac239. Phenotypic analysis of CD4+ T cells demonstrated two patterns of CD4+ T cell depletion, primarily affecting either naïve or memory CD4+ T cells. Progressive decline of total CD4+ T cells was observed only in macaques with naïve CD4+ T cell depletion (ND), though the depletion of memory CD4+ T cells was profound in macaques with memory CD4+ T cell depletion (MD). ND macaques exhibited lower viral load and higher SIV-specific antibody responses and greater B cell activation than MD macaques. Depletion of naïve CD4+ T cells was associated with plasma antibodies autoreactive with CD4+ T cells, increasing numbers of IgG-coated CD4+ T cells, and increased incidence of autoreactive antibodies to platelets (GPIIIa), dsDNA, and phospholipid (aPL). Consistent with a biological role of these antibodies, these latter antibodies were accompanied by clinical features associated with autoimmune disorders, thrombocytopenia, and catastrophic thrombotic events. More importantly for AIDS pathogenesis, the level of autoreactive antibodies significantly correlated with the extent of naïve CD4+ T cell depletion. These results suggest an important role of autoreactive antibodies in the CD4+ T cell decline observed during progression to AIDS., Author Summary Despite intensive study, the mechanisms of CD4+ T cell depletion in human immunodeficiency virus (HIV) infection remain elusive. The identification of the CD4 receptor as the primary receptor for HIV seemed initially to explain the loss of CD4+ T cell lymphocytes in AIDS. However, the number of infected cells at any point in time is insufficient to explain the extent of loss and the slow disease course. While virus-induced cell death might explain early loss of CD4+ T cells, such mechanisms were unlikely to explain the slow depletion over the long period leading to AIDS. Thus, mechanisms related to the intense immune activation, also a hallmark of HIV infection, were proposed. Here, we studied a surrogate model for AIDS, simian immunodeficiency (SIV) infection of rhesus macaques, to explore the mechanisms of this second phase of depletion. We found that animals that exhibited slow, progressive loss of CD4+ T cells had circulating antibodies that reacted with CD4+ T cells and that the levels of these antibodies correlated with the extent of CD4+ T cell depletion. These results suggest that autoimmune destruction of CD4+ T cells represents a valid mechanism to explore in the pathogenesis of CD4+ T cell loss in AIDS.

Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains three binding sites for the transcriptional factor Sp1. In order to investigate the role that the Sp1-binding sites play in regulation of HIV replication, we have introduced a deletion of all three Sp1-binding sites into the LTR of an infectious molecular clone of HIV. Viral stocks have been prepared from this mutant virus, designated dl-Sp, and these stocks have been used to study its replicative ability in human T cells. The dl-Sp virus replicated efficiently in MT4 cells and in phytohemagglutinin-stimulated human peripheral blood lymphocytes, but it replicated poorly and with delayed kinetics in A3.01 (CEM) T cells unless those cells had been treated with the cytokine tumor necrosis factor alpha. Gel retardation assays to study the levels of DNA-binding proteins present in these cells showed that NF-kappa B activity could be detected in the nuclei of MT4 cells but not in A3.01 cells unless they had been treated with tumor necrosis factor alpha. Thus, the presence of NF-kappa B activity appeared to be required for efficient replication of an HIV whose LTR Sp1-binding sites had been deleted. This suggests that NF-kappa B can functionally compensate for Sp1 in activating HIV replication. The HIV LTR is therefore similar to the promoter-enhancer units of other viruses in that it is composed of multiple functional elements that may contribute differently to viral replication depending on the levels of DNA-binding proteins present in the target cells.

Unlike prototypical lentiviruses like visna and caprine arthritis-encephalitis viruses, which are mainly macrophage tropic (M-tropic), primate lentiviruses primarily target CD4 + T lymphocytes. We previously reported that during the late phase of highly pathogenic chimeric simian/human immunodeficiency virus (SHIV) infections of rhesus macaques, when CD4 + T cells have been systemically eliminated, high levels of viremia are maintained from productively infected macrophages. The availability of several different M-tropic SHIVs from such late-stage immunocompromised animals provided the opportunity to assess whether they might contribute to the immune deficiency induced by their T-cell-tropic parental viruses or possibly cause a distinct disease based on their capacity to infect macrophages. Pairs of rhesus monkeys were therefore inoculated intravenously with six different M-tropic SHIV preparations, and their plasma viral RNA loads, circulating lymphocyte subset numbers, and eventual disease outcomes were monitored. Only one of these six M-tropic SHIVs induced any disease; the disease phenotype observed was the typical rapid, complete, and irreversible depletion of CD4 + T cells induced by pathogenic SHIVs. An analysis of two asymptomatic monkeys, previously inoculated with an M-tropic SHIV recovered directly from alveolar macrophages, revealed that this inoculum targeted alveolar macrophages in vivo, compared to a T-cell-tropic virus, yet no clinical disease occurred. Although one isolate did, in fact, induce the prototypical rapid, irreversible, and complete loss of CD4 + T cells, indicating that M-tropism and pathogenicity may not be inversely related, the majority of M-tropic SHIVs induced no clinical disease in immunocompetent macaques.

Rapid progression to disease is observed in patients infected with human immunodeficiency virus (HIV) (9, 13, 30, 38, 40, 60) and macaques experimentally infected with simian immunodeficiency virus (SIV) (15, 18, 21, 22, 24, 31, 44, 47, 52, 64). In both cases, the level of plasma viremia is significantly higher than in conventional progressors, and the cellular and humoral immune responses are very weak and only transiently observed. In this study, as well as prior studies, we defined rapid progressors (RPs) as SIV-inoculated macaques with persistent antigenemia that failed to develop or maintain antibody responses and showed clinical deterioration that necessitated euthanasia before 6 months of inoculation. Recent studies have shown that SIV-infected RP macaques suffer an early massive loss of memory CD4+ T cells, presumably due to virus-induced destruction (24, 31, 34, 37, 44, 45, 52, 61). The loss of memory CD4+ T cells is profound and irreversible in RP macaques, suggesting a strong correlation between the maintenance of CD4+ memory T cells and rapid progression to disease (31, 44, 45, 52). Although these studies of RPs using the SIV model are useful for understanding the mechanism of rapid progression in HIV infection, it is not clear why the depletion of memory CD4+ T cells is so profound in RP macaques and how they develop disease rapidly after the depletion. Recent studies have shown that RP macaques can be distinguished from conventional progressors in terms of pathological manifestations (2, 18, 21, 31). RP macaques uniformly exhibit SIV encephalitis and pneumonia, the presence of multinucleated giant cells, and a predominance of SIV-expressing macrophages in nonlymphoid tissues, such as lung and brain. In contrast, conventional progressor macaques show the pathological features more analogous to AIDS in humans, i.e., the predominance of opportunistic infections. However, the definitive hallmark of rapid progression is the failure to maintain SIV-specific immune responses (18, 21, 64). RP macaques mount an initial humoral and cellular immune response at the appropriate time following infection, but these responses wane rapidly within the first 3 to 4 weeks of infection (21). This immune defect is observed not only in immunity to SIV, but also in immune responses against unrelated antigens, including recall antigens (tetanus toxoid) or new antigen (hepatitis A virus). This profound and global immune failure is consistent with the early loss in T-helper function caused by a massive loss of memory CD4+ T cells (24, 31, 34, 37, 44, 45, 52, 61) and may be critical for the subsequent rapid progression of disease. Despite the lack of immune pressure, the envelope of SIV undergoes unique molecular evolution in RP macaques (8, 31). Sequence analysis of viruses in three SIVsmE543-3-infected RP macaques (8) revealed a unique convergent pattern of substitutions in env, including the loss of a highly conserved potential N-linked glycosylation (PNG) site in the V1-V2 region (N158D/S or S160N/G), substitutions in the V3 analog (P337T/S/H/L and R348W), and substitutions in the highly conserved GDPE motif (G386R and D388N/V). These RP-specific mutations are associated with the acquisition of CD4-independent usage of CCR5 in cell fusion assays (8, 11, 57). In addition, mutational analyses have shown that, like in HIV type 1 (HIV-1) (33, 46), the GDPE motif and/or V3 loop analog of SIV are important for cell tropism and interactions with CD4 and coreceptors (11, 20, 49, 57). These data suggested that RP-specific mutations were selected in RP macaques to adapt to a specific microenvironment in RP macaques through altered cell tropism and receptor usage. In order to study the role of these variants with RP-specific mutations in rapid progression, seven infectious molecular clones were generated from the terminal-phase plasma of an RP macaque (31). We initially predicted that these clones would show an advantage over parental SIV in terms of replication efficacy or cell tropism that would explain the high viral loads in the plasma and tissues of RP macaques. However, clones with RP-specific mutations replicated less efficiently than their parent, SIVsmE543-3, in primary rhesus peripheral blood mononuclear cells and macrophages (31). From these in vitro analyses, it was not clear why RP variants predominated in RP macaques or whether RP variants contributed to the development of rapid disease progression. The goal of the present study was to analyze the biological properties of RP variants in vivo using infectious molecular clones. For this purpose, we chose SIVsmH635FC, since it was closest in terms of sequence to the consensus sequence of plasma virus in the donor animal. Macaques were inoculated with SIVsmH635FC alone or in combination with parental SIVsmE543-3, and virus replication, changes in CD4+ T-cell subsets in blood and mucosal sites, antibody responses, and the stability of RP-specific mutations were evaluated. The coinfected animals allowed us to evaluate the in vivo fitness of SIVsmH635FC relative to that of SIVsmE543-3.

We report here a simple method of directly visualizing in automated DNA sequencing chromatograms DNA methylations of different types including cytosine methylations in Hpa II and dcm sites as well as adenine methylations in dam sites. This is made possible by the observation that the extent of incorporation of fluorescently labeled dideoxynucleotides is influenced by the methylated bases in template DNA. This simple approach involves routine automated DNA sequencing without any prior treatment of DNA specific for detecting DNA methylation.

Several macaques species are used for HIV pathogenesis and vaccine studies, and the characterization of their major histocompatibility complex (MHC) class I genes is required to rigorously evaluate the cellular immune responses induced after immunization and/or infection. In this study, we demonstrate that the gene expressing the Mane-A*06 allele of pig-tailed macaques is an orthologue of the locus encoding the Mamu-A*05 allele family in rhesus macaques. Analysis of the distribution of this locus in a cohort of 63 pig-tailed macaques revealed that it encodes an oligomorphic family of alleles, highly prevalent (90%) in the pig-tailed macaque population. Similarly, this locus was very frequently found (62%) in a cohort of 80 Indian rhesus macaques. An orthologous gene was also detected in cynomolgus monkeys originating from four different geographical locations, but was absent in two African monkey species. Expression analysis in pig-tailed macaques revealed that the Mane-A*06 alleles encoded by this locus are transcribed at 10- to 20-fold lower levels than other MHC-A alleles (Mane-A*03 or Mane-A*10). Despite their conservation and high prevalence among Asian macaque species, the alleles of the Mane-A*06 family and, by extension their orthologues in rhesus and cynomolgus monkeys, may only modestly contribute to cellular immune responses in macaques because of their low level of expression.

Unlike HIV-1 and simian immunodeficiency virus (SIV), which induce a slow, unrelenting loss of immune function spanning several years, highly pathogenic simian–human immunodeficiency viruses (SHIVs) induce a rapid, complete, and irreversible depletion of CD4 + T lymphocytes in rhesus monkeys within weeks of infection, leading to death from immunodeficiency. We recently reported that, because these SHIVs exclusively use the CXCR4 coreceptor for cell entry, they target naïve CD4 + T cells for depletion in infected monkeys, whereas SIVs, which use CCR5, not CXCR4, cause the selective loss of memory CD4 + T lymphocytes in vivo . Here we show both by DNA PCR analyses and infectivity assays, using live sorted CD4 + T lymphocyte subsets, that 30–90% of circulating naïve cells were productively infected by day 10 after inoculation. This result implies that direct cell killing, not bystander apoptosis, is responsible for the massive loss of CD4 + T cells in the X4-tropic SHIV model. Furthermore, we directly demonstrate that >96% of virus producing cells did not express the Ki-67 proliferation marker on day 10 after inoculation using confocal microscopic analysis of lymph nodes samples. This finding is consistent with the prodigious levels of plasma viremia measured during acute X4-tropic SHIV infections of macaques being generated almost entirely by resting naïve CD4 + T cells.

Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques ( Macaca nemestrina ) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4 + -T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4 + -T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4 + -T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques ( P values of + -T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.

The virus infectivity factor (Vif) is a protein encoded by most primate lentiviruses. Recent evidence suggests that HIV-1 Vif reduces the intracellular levels of the host cytidine deaminase APOBEC3G (Apo3G) and inhibits its packaging into virions. These functions of Vif are thought to be species-specific. Accordingly, HIV-1 Vif can target only human Apo3G (hApo3G), whereas, African green monkey simian immunodeficiency virus (SIVagm) Vif can inhibit African green monkey but not human Apo3G. Consistent with this, we found that SIVagm Vif does not affect the stability of exogenously and endogenously expressed hApo3G and does not prevent packaging of exogenous and endogenous hApo3G into SIVagm virions. Nevertheless, SIVagm Vif supported spreading infection of SIVagm virus in the hApo3G-positive human A3.01 T cell line and rescued infectivity of viruses produced from Apo3G-expressing HeLa cells. Sequence analysis verified that SIVagm Vif inhibited the accumulation of hApo3G-induced mutations, suggesting that SIVagm Vif is indeed active in human cells. Our data suggest that SIVagm Vif can inhibit hApo3G activity without inducing its intracellular degradation or preventing its packaging into virions.

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 inex vivoassays, we found that highly pathogenic SHIVDH12Rexclusively uses CXCR4 for infection of rhesus peripheral blood mononuclear cells, whereas SIVmac239and SIVsmE543use CCR5 for entry into the same cells. During the period of peak virus production in SHIVDH12R- or SHIV89.6P-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 cellsin 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.

One of three full-length infectious molecular clones of SHIV DH12R , designated SHIV DH12R-CL-7 and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4 + T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4 + T-cell depletion by SHIV DH12R-CL-7 was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV DH12 . Transfer of the entire SHIV DH12R-CL-7 env gene into the genetic background of nonpathogenic SHIV DH12 failed to confer the rapid CD4 + T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV smE543 for analogous SIV mac239 genes in SHIV DH12R-CL-7 attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4 + T-cell-depleting phenotype exhibited by molecularly cloned SHIV DH12R-CL-7 .

A fraction of simian immunodeficiency virus (SIV)-infected macaques develop rapidly progressive disease in the apparent absence of detectable SIV-specific antibody responses. To characterize the immunopathogenesis of this syndrome, we studied viral load, CD4+T-lymphocyte numbers as well as cellular and humoral immune responses to SIV and other exogenous antigens in four SIVsm-infected rhesus macaques that progressed to AIDS 9 to 16 weeks postinoculation. Each of these animals exhibited high levels of viremia but showed relatively preserved CD4 T lymphocytes in blood and lymphoid tissues at the time of death. Transient SIV-specific antibody responses and cytotoxic T-lymphocyte responses were observed at 2 to 4 weeks postinoculation. Two of the macaques that were immunized sequentially with tetanus toxoid and hepatitis A virus failed to develop antibody to either antigen. These studies show that the SIV-infected rapid progressor macaques initially mounted an appropriate but transient cellular and humoral immune response. The subsequent immune defect in these animals appeared to be global, affecting both cellular and humoral immunity to SIV as well as immune responses against unrelated antigens. The lack of CD4 depletion and loss of humoral and cellular immune responses suggest that their immune defect may be due to an early loss in T helper function.

Passive transfer of high-titered antiviral neutralizing IgG, known to confer sterilizing immunity in pig-tailed monkeys, has been used to determine how soon after virus exposure neutralizing antibodies (NAbs) must be present to block a simian immunodeficiency virus (SIV)/HIV chimeric virus infection. Sterilizing protection was achieved in three of four macaques receiving neutralizing IgG 6 h after intravenous SIV/HIV chimeric virus inoculation as monitored by PCR analyses of and attempted virus isolations from plasma, peripheral blood mononuclear cell, and lymph node specimens. In the fourth animal, the production of progeny virus was suppressed for >4 weeks. A delay in transferring NAbs until 24 h after virus challenge resulted in infection in two of two monkeys. These results suggest that even if a vaccine capable of eliciting broadly reactive NAbs against primary HIV-1 were at hand, the Abs generated must remain at, or rapidly achieve, high levels within a relatively short period after exposure to virus to prevent the establishment of a primate lentivirus infection.

Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 2-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause an irreversible and systemic depletion of CD4+T lymphocytes in macaque monkeys within weeks of inoculation. Nonetheless, the seemingly more aggressive SHIVs have proven to be easier to control by the same vaccine regimens which fail to contain SIV. Because early events during in vivo infections may determine both the pathogenic consequences of the challenge virus and its sensitivity to interventions that prevent disease, we have evaluated the effects of inoculum size and a potent antiretroviral drug on the development of disease in monkeys infected with SHIVDH12R. The results obtained show that in a majority of inoculated animals, suppression of SHIV replication during the first 2 weeks of infection, which prevents complete loss of CD4+T cells, leads to very low to undetectable postpeak viremia and an asymptomatic clinical course for periods up to 4 years.

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala 150 . Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.

The DBA/2 mouseRmcfgene is responsible for in vivo and in vitro resistance to infection by the polytropic mink cell focus-forming (MCF) virus subgroup of murine leukemia viruses (MLVs). Previous studies suggested thatRmcfresistance is mediated by expression of an interfering MCF MLV envelope (Env) gene. To characterize thisenvgene, we examined resistance in crosses betweenRmcfrDBA/2 mice andMus castaneus, a species that lacks endogenous MCFenvsequences. In backcross progeny, inheritance ofRmcfresistance correlated with inheritance of a specific endogenous MCF virusenv-containing 4.6-kbEcoRI fragment. This fragment was present in the DBA/2N substrain withRmcf-mediated resistance but not in virus-susceptible DBA/2J substrain mice. This fragment contains a provirus with a 5′ long terminal repeat and the 5′ half ofenv; thegagandpolgenes have been partially deleted. The Env sequence is identical to that of a highly immunogenic viral glycoprotein expressed in the DBA/2 cell line L5178Y and closely resembles theenvgenes of modified polytropic proviruses. The coding sequence for the full-lengthRmcfEnv surface subunit was amplified from DNAs from virus-resistant backcross mice and was cloned into an expression vector. NIH 3T3 and BALB 3T3 cells stably transfected with this construct showed significant resistance to infection by MCF MLV but not by amphotropic MLV. This study identifies anRmcf-linked MCF provirus and indicates that, like the ecotropic virus resistance geneFv4,Rmcfmay mediate resistance through an interference mechanism.

Previous studies identified three molecular clones of the acutely pathogenic SIVsmPBj strain that varied in terms of relative in vivo pathogenicity. One clone, SIVsmPBj6.6, reproducibly induced a rapidly fatal disease in pigtailed macaques. In contrast, a highly related clone (SIVsmPBj6.9) was only minimally pathogenic in macaques. PBj6.6 and PBj6.9 shared a tyrosine substitution at position 17 in the Nef protein that is a major determinant of virulence but differed at one residue in Vpx (C89R), three residues within the envelope (D119G, R871G, G872R), and a single residue in Nef (F252L). SIVsmPBj6.9 was less efficient in inducing proliferation of resting macaque peripheral blood mononuclear cells in vitro than SIVsmPBj6.6 and exhibited a marked reduction in infectivity relative to SIVsmPBj6.6. Chimeric viruses for each of these variable residues were constructed, and their biologic properties were compared to those of the parental strains. Differences in Vpx and Nef did not alter the basic biologic phenotype of the chimeras. However, the D119G substitution in the envelope of SIVsmPBj6.9 was associated with a marked reduction in the infectivity of this virus relative to SIVsmPBj6.6. An associated processing defect in gp160 of SIVsmPBj6.9 and chimeras expressing the D119G substitution suggests that a reduction in virion envelope incorporation is the mechanistic basis for reduced virion infectivity. In vivo studies revealed that substitution of the PBj6.9 amino acid into PBj6.6 (D119) abrogated the pathogenicity of this previously pathogenic virus. Introduction of the PBj6.9 G119, however, did not confer full virulence to the parental PBj6.9 virus, implicating one or all of the other four substitutions in the virulence of SIVsmPBj6.6.

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies ( Chlorocebus pygerythrus ) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (

A highly pathogenic simian/human immunodeficiency virus (SHIV), SHIV DH12R , isolated from a rhesus macaque that had been treated with anti-human CD8 monoclonal antibody at the time of primary infection with the nonpathogenic, molecularly cloned SHIV DH12 , induced marked and rapid CD4 + T cell loss in all rhesus macaques intravenously inoculated with 1.0 50% tissue culture infective dose (TCID 50 ) to 4.1 × 10 5 TCID 50 s of virus. Animals inoculated with 650 TCID 50 s of SHIV DH12R or more experienced irreversible CD4 + T lymphocyte depletion and developed clinical disease requiring euthanasia between weeks 12 and 23 postinfection. In contrast, the CD4 + T-cell numbers in four of five monkeys receiving 25 TCID 50 s of SHIV DH12R or less stabilized at low levels, and these surviving animals produced antibodies capable of neutralizing SHIV DH12R . In the fifth monkey, no recovery from the CD4 + T cell decline occurred, and the animal had to be euthanized. Viral RNA levels, subsequent to the initial peak of infection but not at peak viremia, correlated with the virus inoculum size and the eventual clinical course. Both initial infection rate constants, k , and decay constants, d , were determined, but only the latter were statistically correlated to clinical outcome. The attenuating effects of reduced inoculum size were also observed when virus was inoculated by the mucosal route. Because the uncloned SHIV DH12R stock possessed the genetic properties of a lentivirus quasispecies, we were able to assess the evolution of the input virus swarm in animals surviving the acute infection by monitoring the emergence of neutralization escape viral variants.

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., Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5α, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission., Author Summary The human immunodeficiency viruses HIV-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and sooty mangabeys (SIVsm), respectively. A related virus, SIVmac, causes AIDS-like pathogenesis in rhesus macaques; like HIV-2, SIVmac is the product of a cross-species jump of SIVsm from sooty mangabeys. The primate TRIM5 gene encodes a factor with potent antiviral activity when tested in the laboratory, and TRIM5 proteins are thought to play a role in restricting the movement of viruses between species in nature. In this study, we show that genetic variation in the TRIM5 gene of rhesus macaques heavily influences the outcome of cross-species transmission of SIVsm and that emergence of SIVmac in rhesus macaques in the 1970s required adaptations to circumvent the genetic barrier imposed by the rhesus macaque TRIM5 gene. Our results confirm the hypothesis that TRIM5 can influence the process of cross-species transmission and emergence of viruses related to HIV-1 and HIV-2 and serve as a striking illustration of how host genes can influence virus evolution.

retroviruses, retroelements and their hosts Meet ing abstracts - A single PDF containing all abstracts in this Supplement is available here . http://www. biomedcentral.co m/content/pdf/17 42-4690-6-S2-inf o.pdf

Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1, lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element(s) was present in the LTR. For example, in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements (and no Sp1 binding sites), a hierarchy of cellular permissivity to virus replication (peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present.

DNA sequences (approximately 11,000 nucleotides) representing the full-length genome of the dengue virus type 4 were cloned. The sequence of the first 2,429 nucleotides at the 5′ terminus which includes the coding region for the structural proteins is presented. The virion structural proteins are encoded in one long open reading frame specifying a polyprotein precursor which is apparently proteolytically cleaved by a mechanism resembling that proposed for expression of structural proteins of other flaviviruses such as yellow fever (YF) and West Nile (WN) viruses. The N terminus for each of the dengue virus structural proteins was tentatively assigned by homology alignment to the corresponding sequence of YF or WN virus. Comparison of sequence homology of structural proteins suggests that dengue virus is more closely related to WN virus than to YF virus or Murray Valley encephalitis virus. Finally, analysis of the extreme 5′- and 3′-terminal nucleotides of the dengue virus genome revealed sequences that may be involved in transcription, replication, and packaging of viral RNA.

Complete sequences for the intergenic regions of the genome of human respiratory syncytial virus were obtained by dideoxynucleotide sequencing using synthetic oligonucleotides. These experiments established that the 10 respiratory syncytial viral genes are arranged, without additional intervening genes, in the order 3' 1C-1B-N-P-M-1A-G-F-22K-L 5'. For the first nine genes, the exact gene boundaries were identified by comparison of the genomic sequences with previously determined mRNA sequences. The intergenic regions varied in length from 1 to 52 nucleotides and lacked any obvious conserved features of primary or secondary structure except that each sequence ended (3' to 5') with an adenosine residue. The exact start site of the 10th gene, the L gene, was not determined. However, RNA blot hybridization using a synthetic oligonucleotide designed from the genomic sequence mapped the L gene to within 54 nucleotides of the end of the penultimate 22K gene. The lack of conservation of chain length and nucleotide sequence for the respiratory syncytial viral intergenic regions, together with the complexity of the genetic map, contrasts with previous observations for other nonsegmented negative-strand viruses.

We recently cloned a full-length DNA copy of the dengue type 4 virus genome. Analysis of the 5' terminal nucleotide sequence suggested that the three-virion structural proteins are synthesized by proteolytic cleavage of a polyprotein precursor which is encoded in one open reading frame. We now present the remaining sequence of the dengue type 4 virus genome which codes for the nonstructural proteins. The entire genome, which is 10,644 nucleotides in length, contains one long open reading frame which codes for a single large polyprotein 3386 amino acids in length. Alignment of the dengue nonstructural protein sequence with that of other flaviviruses, including yellow fever and West Nile viruses, revealed that significant homology exists throughout the entire nonstructural region of the dengue genome and this allowed tentative assignment of individual nonstructural proteins in the following order: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5-COOH. Processing of the nonstructural proteins appears to involve two types of proteolytic cleavage: the first occurs after a long hydrophobic signal sequence and the second occurs at a junction between two basic amino acids and a small polar amino acid. A notable exception is the cleavage at the N-terminus of the dengue NS3 which may take place at the junction between Gln-Arg and Ser. Comparative analysis suggests that dengue NS3 and NS5 may be involved in enzymatic activities related to viral replication and/or transcription. Putative nonstructural proteins NS2a, NS2b, NS4a, and NS4b are extremely hydrophobic, suggesting that these proteins are most likely associated with cellular membranes.

Reassortant influenza A viruses were produced by mating an avian virus (A/Mallard/NY/78, A/Mallard/Alberta/78, or A/Pintail/Alberta/79) with a wild-type human influenza A virus. From each mating a reassortant virus was obtained that contained the genes coding for the hemagglutinin and neuraminidase surface antigens of the human influenza A wild-type virus and the six other RNA segments ("internal genes") of the avian influenza A virus parent. The avian-human reassortant influenza viruses produced resembled their avian virus parent in that they produced plaques on MDCK monolayers at 42 C, a temperature restrictive for the human influenza viruses. In the trachea of squirrel monkeys, each avian-human reassortant influenza virus was as restricted in its replication as was its avian influenza virus parent. Thus, one or more of the six internal genes of each avian parent virus was responsible for restriction of the reassortant virus in monkeys. The A/Washington/80 X A/Mallard/NY/78 reassortant virus retained its phenotype of restricted replication in monkeys after five serial passages in vivo. It also failed to transmit to cagemates or induce resistance to wild-type virus challenge, and it did not initiate a systemic or enteric infection. These findings form the basis for evaluation of these attenuated avian-human reassortant influenza A viruses as live attenuated vaccines for humans.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. In the case of HIV, the antiviral activity of APO3G is counteracted by the viral Vif protein. Monocyte-derived macrophages (MDM) are terminally differentiated, non-dividing cells susceptible to HIV infection. Human MDM are known to express APO3G and HIV replication in these cells is dependent on Vif. Here we analyzed the correlation between HIV-1 replication and APO3G expression in MDM. Replication of wild type HIV-1 induced a gradual 4-5-fold reduction in APO3G expression. The efficiency of APO3G downregulation correlated with the efficiency of virus replication. Interestingly, despite downregulation of APO3G, the relative infectivity of viruses rapidly declined during the course of infection and was already reduced approximately 90% prior to peak virus production. Cell-free virus preparations showed increased levels of a 41 kDa MA-CA processing intermediate. Sequence analysis around the MA-CA cleavage site and the protease and LTR regions did not reveal deaminase-induced hypermutation of the viral genome, suggesting that APO3G activity is not responsible for the incomplete Gag processing. Thus, the loss of infectivity of HIV-1 viruses produced from long-term infected primary macrophages is due to an APO3G-independent mechanism.