Ubiquitin Is Covalently Attached to the p6Gag Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6^Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12^Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6^Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.     

表达猴免疫缺陷病毒Env蛋白的重组鸡痘病毒的构建和筛选

目的:构建并筛选表达猴免疫缺陷病毒(SIV)Env蛋白的重组鸡痘病毒(FPV),并对其进行鉴定。方法:设计引物,通过PCR技术扩增SIV env基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的FPV穿梭载体pTKET中,获得重组质粒pTKET-SIV env,然后将其与FPV282E4株共转染原代鸡胚成纤维细胞进行同源重组,以增强型绿色荧光蛋白为筛选标记,通过噬斑筛选获得重组病毒,应用PCR、RT-PCR、Western印迹对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组FPV基因组中,RT-PCR、Western印迹结果表明SIV Env蛋白在感染细胞内表达且具有抗原性;连续传代20次,PCR、RT-PCR、Western印迹均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,且病毒已经纯化。结论:获得表达SIV Env蛋白的重组FPV,为进一步免疫试验研究奠定了基础。     

Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.     

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.     

Simian Virus 40 Chromatin Interaction with the Capsid Proteins

Abstract

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40°C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100–160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100–160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40°C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.     

Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4⁺ and CD8⁺ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.Simian immunodeficiency virus (SIV) infection in African green monkeys (AGM) is nonpathogenic, even though it is characterized by plasma viral load (PVL) levels similar to those found during acute and chronic pathogenic infection of humans with human immunodeficiency virus type 1 and macaques with SIVmac (14). This feature is shared with other African nonhuman primates, such as sooty mangabeys (SM) and mandrills (19, 20). SIV-infected AGMs also display high viral loads in the gastrointestinal mucosa (11), a transient decline of circulating CD4⁺ T cells during acute infection (13), and longer-lasting CD4⁺ T-cell depletion in the intestinal lamina propia (10). Concomitant with the peak viral load during acute infection, SIVagm-infected AGMs display transient increases of CD4⁺ and CD8⁺ T cells expressing activation, and proliferation markers, such as MHC-II DR and Ki-67 (4, 13), and anti-SIVagm antibodies (Ab) are induced with kinetics similar to those found in SIVmac infection (5). Interestingly, however, the Ab response against Gag p27 is weak, if present at all (1, 2, 12, 15, 17, 18). This observation is surprising since, in the context of human immunodeficiency virus type 1 and SIVmac infections, Ab responses to Gag p27 are usually quite strong. Weak or low reactivity to Gag p27 has also been observed in some other natural SIV infections (7, 8, 20) but not in all of them (21). We wondered whether such a selective lack of Ab reactivity in the SIV-infected AGM might be related to a lack of Gag p27-specific T cells. With this hypothesis in mind, we first confirmed and extended the studies of humoral responses against Gag p27 by characterizing the antigen-specific immunoglobulin G (IgG) responses and mid-point titers against total SIVagm antigens (SIVagm virions) and recombinant Gag p27 (rP27; SIVagm) in naturally and experimentally SIVagm-infected AGMs. Second, we searched for the presence of Gag p27-specific T-cell responses in SIVagm infection by analyzing the CD4⁺ and CD8⁺ T-cell responses specific for Gag p27 and other SIVagm proteins in blood and lymph nodes (LNs) of acutely and chronically infected animals.Humoral responses against SIV were analyzed in 50 wild-born AGMs (Chlorocebus sabaeus) and 17 rhesus macaques (RMs). The animals were housed at the Institut Pasteur in Dakar, Senegal, and the California National Primate Research Center, Davis, CA, respectively, according to institutional and national guidelines. RMs were either noninfected (n = 5) or intravenously infected with SIVmac251 (n = 12). AGMs were noninfected (n = 23), naturally infected (n = 17), or intravenously infected with wild-type SIVagm.sab92018 (n = 10) (5, 9). IgG titers against SIVagm.sab92018 virions or rP27 were determined by an enzyme-linked immunosorbent assay (ELISA) using monkey anti-IgG as secondary Ab (Fig. 1A and B). The virions had been purified by ultracentrifugation on an iodixanol cushion from cell-free supernatants of SIVagm.sab92018-infected SupT1 cells. The His-tagged rP27 was constructed using DNA from gut cells of an SIVagm.sab92018-infected AGM 96011 (11). A Gag p27 PCR product was subcloned into pET-14b, and the recombinant protein was produced in Escherichia coli BL21(DE3)(pLysS) and purified on nitrilotriacetic acid columns. SIV-infected macaques showed high IgG titers cross-reacting with both SIVagm virions (Fig. 1A and B, left panels) and rP27 (Fig. 1A and B, right panels). In contrast, only 2 out of 27 SIV-infected AGMs showed detectable IgG responses against rP27 (Fig. 1A and B, right panels), while 21 out of 27 displayed significant responses against SIVagm virions (Fig. 1A and B, left panels). Two AGMs out of 23 from the negative control group showed weak responses at the limit of detection against SIVagm and two against rP27, suggesting a natural response against SIVagm proteins, cross-reactivity with unknown pathogens, maternal Ab, or recent SIV infection. Of note, the titers against whole SIV in the infected monkeys were higher in macaques than in AGMs, which may be due to a lack of anti-p27 Ab in most AGMs.Open in a separate windowFIG. 1.Cross-sectional analysis of IgG Ab responses against SIVagm or Gag p27 in SIV-infected AGMs and RMs. (A and B) Cross-sectional analysis by ELISA. IgG Ab against SIVagm.sab₉₂₀₁₈ virions or recombinant p27-Gag antigens were determined in SIV-negative (Rh SIV−) and chronically SIVmac₂₅₁-infected (Rh SIV+) RMs and in SIV-negative and chronically SIVagm-infected AGMs that were either naturally (AGM Nat SIV+) or experimentally (AGM Exp SIV+) infected with SIVagm.sab₉₂₀₁₈. Ab titers were calculated for each animal by limited dilution of plasma on coated ELISA plates with 5 μg/ml of (p27 equivalent) virions (left) or 1 μg/ml of the monomeric recombinant protein (rP27) (right). IgG detection by ELISA displayed a high background for rP27, especially at the highest plasma concentration (e.g., 1/100 and 1/400 plasma dilution) in SIV-negative RMs and AGMs. To discriminate between positive responses and background, calculated dose-response curves were compared to theoretical sigmoid-dose response curves corresponding to the 95% confidence interval of SIV-negative animals. By convention, responses were considered background when sigmoid dose-response curves were graphically within the 95% confidence interval of SIV-negative animals and when the calculated negative log 50% effective concentration (EC₅₀) was lower than the top theoretical sigmoid dose-response curve from SIV-negative animals (corresponding to a threshold of negative log EC₅₀ of 2.8). (A) Results (optical density at 450 nm [OD₄₅₀]) are represented for both virions (left) and rP27 (right) over plasma dilution (log₁₀) on a per animal basis (data points) and for each group (lines). Lines represent the sigmoid dose-response curves for each group (Prism 4; Graphpad). (B) Mid-point IgG titers were determined for each animal from individual sigmoid dose-response curves, and presented as the log₁₀ value from the reciprocal of the effective concentration that corresponds to 50% response between minimum and maximum OD₄₅₀ (negative log EC₅₀). Horizontal bars represent the median mid-point titer per each group. Mann-Whitney nonparametric tests were applied for statistical analysis (n.s., nonsignificant, with P values of >0.1) (C) Cross-sectional analysis of Ab against SIVagm proteins by Western blot analysis using denatured SIVagm.sab₉₂₀₁₈. For the positive controls on the left, we used sera from an SIVmac₂₅₁-infected macaque and a SIVagm.sab₉₂₀₁₈-infected AGM. Development times and reagents were identical for all Western blots. Mo, months of infection; y, years of infection; C−, negative control; C+, positive control.The study of IgGs by Western blot analysis using denatured SIVagm.sab92018 virions showed no or weak anti-Gag responses in SIV-infected AGMs, yet the anti-Env responses were often strong (Fig. ​(Fig.1C).1C). In contrast, SIV-infected macaques showed a dominant IgG cross-reactive response against the SIVagm Gag p27 protein. Even if responses in AGMs were detected more frequently with the Western blot analyses than with the ELISAs, these responses were different in magnitude and considerably weaker than those in macaques.To compare B- and T-cell responses over time, five simian T-cell leukemia virus-seronegative AGMs were infected with SIVagm.sab92018, and the animals were followed longitudinally during the acute and postacute phases of infection until day 90 postinfection (p.i.). Sequential blood samples were collected and biopsies of auxiliary and inguinal LNs were performed on day −5 and at three times p.i. (days 14, 43, and 62). PVL was measured by real-time PCR (5). Since we searched for Gag p27-specific responses, we also quantified Gag p27 antigen in the plasma (SIV p27 antigen assay; Coulter, Miami, FL). Viral RNA and p27 antigenemia peaks were observed between days 7 and 14 p.i. (Fig. 2A and B, respectively). The Gag p27 levels were variable among the animals but in a range similar to those reported previously in AGMs and macaques (3, 5). As has also been observed in SIVmac infection (except for rapid progressors), plasma Gag p27 levels fell below the detection level in the postacute phase (i.e., after day 28 p.i.) (Fig. ​(Fig.2B2B and data not shown). There were significant increases in circulating CD8⁺ DR⁺ T cells at days 7 and 14 p.i. and in CD8⁺ Ki-67⁺ T cells at days 14 and 28 p.i. (Fig. 2C and D, left panels). After day 28 p.i., the percentages were no longer statistically different from baseline levels. In LN cells (LNCs), the percentage of CD8⁺ Ki-67⁺ T cells rose from 3.1% ± 1.1% before infection to 6.1% ± 0.3% at day 62 p.i., but the difference was not statistically significant (Fig. ​(Fig.2D,2D, right panel). The levels of blood CD4⁺ DR⁺ Ki-67⁺, CD8⁺ DR⁺ Ki-67⁺, CD8⁺ Ki-67⁺ T cells, and LNC CD8⁺ Ki-67⁺ T cells were positively correlated with viremia (P values of 0.002 for DR⁺ cells and P values of <0.02 for Ki-67⁺ cells). Altogether, these results confirm previous data showing early, transient T-cell activation in the peripheral blood of SIVagm-infected AGMs (13).Open in a separate windowFIG. 2.Plasma viremia and T-cell activation in blood and LNs of five longitudinally followed SIVagm.sab₉₂₀₁₈-infected African green monkeys. (A) SIVagm.sab RNA copy numbers in plasma. (B) Plasma Gag p27 concentrations. (C) Percentages of MHC-II DR-positive CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. (D) Percentages of Ki-67⁺ CD4⁺ (•) and CD8⁺ (○) T cells within, respectively, total CD4⁺ and CD8⁺ T cells from PBMCs and LNCs. Results are shown as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to levels before infection (P < 0.05).We next looked for the presence of Ab responses against rP27 in these animals. No Ab were detected before infection. After infection, all five AGMs developed anti-SIVagm IgGs within 4 to 9 weeks p.i., with AGM 02001 showing the fastest response (Fig. ​(Fig.3A).3A). While the humoral responses against whole virions were significant (Fig. ​(Fig.3B),3B), the anti-rP27 responses were below the threshold for positivity (Fig. ​(Fig.3B),3B), with the exception of one animal (AGM 02001). The anti-rP27 response in this animal was only transient since it was no longer detectable at week 75 p.i., in contrast to the anti-SIV Ab that were sustained (Fig. ​(Fig.3B3B and data not shown).Open in a separate windowFIG. 3.Longitudinal analysis of IgG titers and T-cell proliferative responses against SIVagm and Gag p27 in five AGMs experimentally infected with SIVagm.sab₉₂₀₁₈. (A and B) Ab responses were analyzed by ELISA. (A) IgG dose-response curves against SIVagm (top) and rP27 (bottom) are shown over time (week −1 to week 24 p.i.). O.D.450, optical density at 450 nm. (B) Mid-point titers were calculated as described in the legend to Fig. ​Fig.1A.1A. Continuous lines correspond to median titers from all five animals. Red, anti-SIVagm IgGs; green, anti-p27 IgGs. (C) Proliferative responses of CD4⁺ and CD8⁺ T cells were assessed by flow cytometry using carboxy fluorescein succinimidyl ester staining (CFSE). CD4⁺ and CD8⁺ T-cell responses in PBMCs (left) and LNCs (right) after stimulation with peptide pools (Gag without P27, P27, and Tat) and Gag rP27 are shown for each animal. All data are reported after background subtraction. Results are presented in columns as the mean ± the standard error of the mean. Asterisks indicate statistically significant differences compared to individual values before infection (P < 0.05).We next searched for T-cell responses against Gag p27 compared to other SIVagm antigens in these animals. Gag p27 epitopes were presented in the following two ways: in the context of rP27 and as synthetic peptides. The peptide pools (comprised of overlapping 15-mers) spanned the following SIVagm proteins: Gag p27, Gag without p27, Env, and Tat. The amino acid sequences of the Gag and Env peptides corresponded to the autologous wild-type SIVagm.sab92018 sequence, and those of the Tat peptides corresponded to an SIVagm.sab consensus sequence. The latter was determined using Tat sequences of other SIVagm viruses from Senegal that are available in the databases (SIVagm.sab1c, SIVagm.sabD42, and SIVagm.sabD30). We measured T-cell responses by investigating the antigen-induced proliferation. T cells from blood (peripheral blood mononuclear cells [PBMCs]) and LNs were analyzed. All assays were performed with fresh cells that were stimulated with 10 μg/ml of Gag rP27 and 5 μg/ml of peptides over a period of 4 days. Dead cells were gated out using 7-amino-actinomycin D, and dividing (CFSE^low) cells were analyzed after stimulation with medium alone, SIV antigens, or concanavalin A as a positive control. We detected significant Gag p27-specific proliferative responses for CD8⁺ T cells in PBMCs and for CD4⁺ and CD8⁺ T cells in LNCs (Fig. ​(Fig.3C).3C). The animal with the detectable anti-p27 Ab (AGM 02001) did not show stronger p27-specific T-cell responses than the other animals. Thus, all SIV-infected AGMs were able to mount a proliferative T-cell response against p27, while anti-p27 IgGs were lacking in four of the animals. However, the SIVagm-specific T-cell responses were detected at only a few time points p.i.We then analyzed the T-cell responses in the chronic phase of AGMs naturally and experimentally infected with SIVagm.sab92018. PVL, peripheral blood cell counts (CD4⁺ and CD8⁺ T cells; CD20⁺ B cells), and immune activation (Ki-67⁺ CD4⁺ and CD8⁺ T cells) were similar in naturally infected and in experimentally infected AGMs (Fig. ​(Fig.4A).4A). As expected, cell counts and immune activation levels were also not different from SIV-negative AGMs (Fig. ​(Fig.4A).4A). Again, we measured SIV-specific responses first by a proliferation assay (Fig. ​(Fig.4B).4B). One out of five animals tested had a proliferative SIV-specific CD4⁺ T-cell response (against Gag without p27, P27, rP27, Env GP120, and Tat), and two animals had a CD8⁺ T-cell response (against P27 in both animals and against Env GP120 and Tat in one). Two animals (one naturally infected and one experimentally infected with SIVagm.sab92018) did not show any detectable antigen-specific proliferative CD4⁺ or CD8⁺ T-cell response.Open in a separate windowFIG. 4.Immune parameters and SIVagm-specific proliferative and cytokine T-cell responses in chronically infected AGMs. (A) Cell counts (CD4⁺ and CD8⁺ T cells; B cells) and immune activation levels (percent of Ki-67⁺ in CD4⁺ and CD8⁺ T cells) in AGMs (n = 4) naturally infected with SIVagm (Nat SIV⁺) and AGMs (n = 6) experimentally infected with SIVagm.sab₉₂₀₁₈ (Exp SIV⁺) compared to uninfected AGMs (n = 10) (SIV⁻). PVL, if known, is indicated. Green, blue, and orange symbols correspond, respectively, to noninfected, naturally infected, and experimentally infected AGMs. (B) Proliferative response to SIVagm antigens in chronically infected AGMs (n = 5) compared to those in uninfected AGMs (n = 3). PBMCs were stimulated with the same antigens as those described in the legend to Fig. ​Fig.3.3. (C) Analysis of cytokine responses (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) by SIVagm-specific T cells. ConA was used as a positive control. Representative results from a single animal are shown here. (D) Cumulative values of SIVagm-specific TNF-α and IFN-γ responses in chronically infected animals. The responses to SIVagm antigens were analyzed in peripheral blood specimens of 4 naturally and 5 experimentally infected AGMs as well as 10 uninfected AGMs. The data are reported after background subtraction corresponding to the subtraction of the frequency of positive events from the unstimulated samples to the frequency of positive events from the antigen-specific stimulation. Proliferative T-cell responses and cytokine T-cell responses in SIV-infected AGMs were defined as positive when higher than 3 standard deviations above the mean responses for uninfected animals. Freq, frequency; w/o, without.These results were extended to an analysis of SIV-specific T-cell cytokine responses, e.g., the production of IFN-γ and TNF-α in nine chronically infected compared to 10 noninfected AGMs (Fig. 4C and D). Fresh cells were stimulated for 8 h with the antigens described above. SIV-specific cytokine responses were detected in CD8⁺ but not in CD4⁺ T cells. Seven animals out of nine showed a response against at least one antigen. The two animals showing no response were among the four naturally infected animals tested. We therefore cannot exclude that the absence of response in these two animals is due to the presence of highly divergent viruses. However, a precise epitope mapping in SIVagm sequences would be necessary to confirm this. In those animals showing a SIVagm-specific cytokine T-cell response, the responses were directed against Gag p27 (four out of nine animals), other Gag proteins than p27 (two out of nine animals), and Env GP120 (four out of nine animals). In the experimentally infected animals, we might have underestimated the responses against Tat compared to Gag and Env antigens, since the Tat peptides corresponded to an SIVagm.sab consensus sequence and not to the autologous virus (SIVagm.sab92018). There was no correlation between the magnitude or breadth of SIV-specific T-cell responses and immune activation or PVL.Altogether, our study demonstrates that AGMs can mount T-cell proliferative and cytokine responses against Gag p27. The T-cell response was variable among the animals. In general, it appeared moderate, comparable to chronically SIV-infected RMs (9). Of note, T-cell responses were not consistently detected at all time points and not in all animals. We cannot exclude the possibility that we underestimated the magnitude of the cytokine responses. For instance, we did not costimulate the cells during the assays. However, cytokine responses were also variable in vervet AGMs, with a trend for reduced levels compared to those for RMs, even when more-sensitive assays were used (23). In SM, the responses were also reported to be not stronger than in RMs. This is in line with the lack of efficient control of viral replication in natural hosts (6, 22).In our study, we show that IgG responses against Gag p27 are either lacking, weak, or transient, while Ab against other SIVagm proteins are present. The mechanisms underlying this selective lack of Gag p27 Ab responses are unclear. It could be related to moderate and/or dysfunctional CD4⁺ T-cell responses and/or due to an unknown suppressive regulatory mechanism. SIV-specific T-cell cytokine responses were indeed principally found at the CD8⁺ T-cell level. This was also reported in SIVsm-infected SM (6, 22). Here, we also searched for SIVagm Gag p27-specific proliferative responses. Interestingly, they were detected for CD4⁺ T cells, indicating the presence of p27-specific CD4⁺ memory cells in AGMs. Moreover, AGMs can potentially mount a strong and sustained anti-Gag p27 humoral response, when appropriately immunized (D. Favre et al., unpublished data). This suggests that there is neither a central B-cell tolerance against p27 Gag protein in AGMs nor an inherent inability for CD4⁺ T cells to provide helper B-cell functions. The transient nature of anti-p27 Ab in one animal would be in favor of regulatory mechanisms, but that needs to be confirmed. Another explanation could be that AGMs are able to mount Ab responses against some p27 epitopes but not to those exposed by the native protein, which would explain why we and others detect more frequently humoral responses in Western blot analysis than in ELISAs (16).In conclusion, we characterized the IgG responses against SIVagm and confirmed a lower humoral response against p27 than in RMs. Moreover, our study reveals that cytokine and proliferative T-cell responses against SIVagm Gag p27 are detectable in AGMs. Thus, the reduced ability of the AGM to produce Ab against Gag p27 p.i. is not related to a lack of Gag p27-specific T cells.     

Evolution of a Simian Immunodeficiency Virus Pathogen

Analysis of disease induction by simian immunodeficiency viruses (SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since they failed to induce disease within 1 year postinoculation in any inoculated animal. Here we report the natural history of infection with BK28 and BK44 in inoculated rhesus macaques and efforts to increase the pathogenicity of BK28 through genetic manipulation and in vivo passage. BK44 infection resulted in no disease in four animals infected for more than 7 years, whereas BK28 induced disease in less than half of animals monitored for up to 7 years. Elongation of the BK28 transmembrane protein (TM) coding sequence truncated by prior passage in human cells marginally increased pathogenicity, with two of four animals dying in the third year and one dying in the seventh year of infection. Modification of the BK28 long terminal repeat to include four consensus nuclear factor SP1 and two consensus NF-κB binding sites enhanced early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from immunodeficiency disease (1.5 years after infection) resulted in a consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains identified to date. To determine whether the diverse viral quasispecies that evolved during in vivo passage was required for pathogenicity or whether a more virulent virus variant had evolved, we generated a molecular clone composed of the 3′ half of the viral genome derived from the in vivo-passaged virus (H824) fused with the 5′ half of the BK28 genome. Kinetics of disease induction with this cloned virus (BK28/H824) were similar to those with the in vivo-passaged virus, with four of five animals surviving less than 1.7 years. Thus, evolution of variants with enhanced pathogenicity can account for the increased pathogenicity of this SIV strain. The genetic changes responsible for this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have been multiple and dispersed, including elongation of the TM and Nef coding sequences; changes in RNA splice donor and acceptor sites, TATA box sites, and Sp1 sites; multiple changes in the V2 region of SU, including a consensus neutralization epitope; and five new N-linked glycosylation sites in SU.     

Specific Interactions between Retrovirus Env and Gag Proteins in Rat Neurons

In this work we have studied the intracellular localization properties of the Gag and Env proteins of Moloney murine leukemia virus (MLV) and human immunodeficiency virus (HIV) in dorsal root ganglion (DRG) neurons of rat. These neurons form thick bundles of axons, which facilitates protein localization studies by immunofluorescence analyses. When such neuron cultures were infected with recombinant Semliki Forest virus particles carrying the gag genes of either retrovirus, the expressed Gag proteins were localized to both the somatic and the axonal regions of the DRG neurons. In contrast, the Env proteins were confined only to the somatic region. When the Gag and Env proteins were coexpressed, the Gag proteins were also excluded from the axons. This effect of the Env proteins was shown to be dependent on the concentration of the Gag proteins in the neuron and also to be specific for homologous pairs of retrovirus proteins. Therefore, the results suggest that there are specific interactions between the Env and the Gag proteins of MLV and HIV in the DRG neurons.     

Distinct Evolutionary Pressures Underlie Diversity in Simian Immunodeficiency Virus and Human Immunodeficiency Virus Lineages

Simian immunodeficiency virus (SIV) infection of rhesus macaques causes immune depletion and disease closely resembling human AIDS and is well recognized as the most relevant animal model for the human disease. Experimental investigations of viral pathogenesis and vaccine protection primarily involve a limited set of related viruses originating in sooty mangabeys (SIVsmm). The diversity of human immunodeficiency virus type 1 (HIV-1) has evolved in humans in about a century; in contrast, SIV isolates used in the macaque model evolved in sooty mangabeys over millennia. To investigate the possible consequences of such different evolutionary histories for selection pressures and observed diversity in SIVsmm and HIV-1, we isolated, sequenced, and analyzed 20 independent isolates of SIVsmm, including representatives of 7 distinct clades of viruses isolated from natural infection. We found SIVsmm diversity to be lower overall than HIV-1 M group diversity. Reduced positive selection (i.e., less diversifying evolution) was evident in extended regions of SIVsmm proteins, most notably in Gag p27 and Env gp120. In addition, the relative diversities of proteins in the two lineages were distinct: SIVsmm Env and Gag were much less diverse than their HIV-1 counterparts. This may be explained by lower SIV-directed immune activity in mangabeys relative to HIV-1-directed immunity in humans. These findings add an additional layer of complexity to the interpretation and, potentially, to the predictive utility of the SIV/macaque model, and they highlight the unique features of human and simian lentiviral evolution that inform studies of pathogenesis and strategies for AIDS vaccine design.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Common Themes of Antibody Maturation to Simian Immunodeficiency Virus, Simian-Human Immunodeficiency Virus, and Human Immunodeficiency Virus Type 1 Infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.     

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.