Requirement for CD4+ T Cells in the Friend Murine Retrovirus Neutralizing Antibody Response: Evidence for Functional T Cells in Genetic Low-Recovery Mice

Recovery from infection with the Friend murine leukemia retrovirus complex (FV) requires T-helper cells and cytotoxic T cells as well as neutralizing antibodies. Several host genes, including genes of the major histocompatibility complex (H-2) and an H-2-unlinked gene, Rfv-3, influence these FV-specific immune responses. (B10.A × A/Wy)F₁ mice, which have the H-2^a/a Rfv-3^r/s genotype, fail to mount a detectable FV-specific T-cell proliferative response but nevertheless produce FV-specific neutralizing immunoglobulin M (IgM) antibodies and can eliminate FV viremia. Thus, this IgM response, primarily influenced by the Rfv-3 gene, may be T-cell independent. To test this idea, mice were depleted of either CD4⁺ or CD8⁺ T-cell populations in vivo and were monitored for the effect on the neutralizing antibody response following FV infection. Surprisingly, mice in which CD4⁺ cells were depleted showed undetectable FV-neutralizing antibody responses and high viremia levels compared to nondepleted or CD8-depleted animals. In addition to knocking out the FV antibody response, CD4⁺ T-cell depletion reduced survival time significantly, further indicating the importance of CD4⁺ T cells. These studies revealed the first evidence for a functional T-cell response following FV infection in these low-recovery mice and showed that CD4⁺ T-helper cells are required for the Rfv-3-controlled FV antibody response.     

Long-Lasting Protective Antiviral Immunity Induced by Passive Immunotherapies Requires both Neutralizing and Effector Functions of the Administered Monoclonal Antibody

Using FrCas^E retrovirus-infected newborn mice as a model system, we have shown recently that a long-lasting antiviral immune response essential for healthy survival emerges after a short treatment with a neutralizing (667) IgG2a isotype monoclonal antibody (MAb). This suggested that the mobilization of adaptive immunity by administered MAbs is key for the success in the long term for the MAb-based passive immunotherapy of chronic viral infections. We have addressed here whether the anti-FrCas^E protective endogenous immunity is the mere consequence of viral propagation blunting, which would simply give time to the immune system to react, and/or to actual immunomodulation by the MAb during the treatment. To this aim, we have compared viral replication, disease progression, and antiviral immune responses between different groups of infected mice: (i) mice treated with either the 667 MAb, its F(ab′)₂ fragment, or an IgM (672) with epitopic specificity similar to that of 667 but displaying different effector functions, and (ii) mice receiving no treatment but infected with a low viral inoculum reproducing the initial viral expansion observed in their infected/667 MAb-treated counterparts. Our data show that the reduction of FrCas^E propagation is insufficient on its own to induce protective immunity and support a direct immunomodulatory action of the 667 MAb. Interestingly, they also point to sequential actions of the administered MAb. In a first step, viral propagation is exclusively controlled by 667 neutralizing activity, and in a second one, this action is complemented by FcγR-binding-dependent mechanisms, which most likely combine infected cell cytolysis and the modulation of the antiviral endogenous immune response. Such complementary effects of administered MAbs must be taken into consideration for the improvement of future antiviral MAb-based immunotherapies.Although monoclonal antibodies (MAbs) principally have been considered for anticancer applications heretofore (62, 64), they now are increasingly being considered to treat severe acute and chronic viral infections (43, 63, 83). The best-studied antiviral MAbs are (i) pavalizumab, a humanized anti-respiratory syncytial virus (RSV) MAb approved by the FDA in 1998 for treating severe lower-respiratory-tract diseases in infants (45); (ii) several anti-human immunodeficiency virus (HIV) MAbs, which have been used in macaque preclinical infection models and in several human trials (4, 5, 19, 27-30, 32, 42, 50, 55, 57, 76-79); and (iii) a few anti-hepatitis C virus (HCV) MAbs, some of which currently are being tested in humans (9, 22, 40). However, other MAbs, some of them of human origin, also have been generated against other human viruses in recent years. Among them are antibodies against Ebola virus (75), West Nile virus (WNV) (48, 53, 54), cytomegalovirus (CMV) (11), avian and human influenza viruses (59, 60, 73, 74), severe acute respiratory syndrome coronavirus (SARS CoV) (81), hepatitis B virus (HBV) (31, 35), Hanta virus (80, 82), and Nipah virus (80, 82). These antiviral MAbs all have been selected on the basis of their neutralizing activity and the possibility that they interfere with the antiviral immune response of treated hosts, because their effector functions have been considered surprisingly little so far. Addressing this question in clinical settings currently is not possible for a variety of reasons that include ethical, technical, and cost concerns. Therefore, we have turned to the neonatal infection of mice by the lethal FrCas^E retrovirus as a model system. This model allowed us to show that a very short immunotherapy by a neutralizing MAb of the IgG2a isotype (667 MAb) can permit, in addition to an immediate direct effect on the viral load, the mounting of a long-lasting endogenous antiviral immunity, which is essential for viral control and healthy survival (23-25). Because of the broad therapeutic perspectives opened by this observation, it now is essential to elucidate the molecular and cellular mechanisms underlying this effect.FrCas^E is a simple chimeric mouse retrovirus in which the env gene of the leukemogenic Friend murine leukemia virus (F-MuLV) was replaced by that of the neurodegeneration-inducing CasBr retrovirus (58). When 5 × 10⁴ infectious particles are inoculated into newborn mice under the age of 5 to 6 days, FrCas^E can enter the central nervous system (CNS) and induces a neurodegeneration fatal within 1 to 2 months with 100% incidence (15, 23, 41, 58). However, upon infection at a later time, FrCas^E can no longer enter the CNS. Instead, it replicates only in the periphery and gives rise to a fatal erythroleukemia preceded by spleen enlargement and a dramatic drop of the hematocrit. Erythroleukemia incidence and incubation period, however, are variable, depending on the inoculum and the date of infection (46).667 is an IgG2a/κ (44) directed to the main viral receptor-binding site of CasBr Env (16). It displays both in vitro (44) and in vivo (56) neutralizing activities. When rapidly (<2 days) administered for a few days to neonatally FrCas^E-infected pups, viral propagation is rapidly blunted, which prevents virus entry in the brain and subsequent neurodegeneration (23). Moreover, all 667-treated mice develop a strong, long-lasting antiviral immune response, which is necessary for them to survive healthy and with no sign of neurodegeneration or of erythroleukemia (23-25) and to resist viral challenges carried out as long as 14 months after first infection (23). Protective antiviral immunity is of a typical TH1 type with humoral and cytotoxic T-cell (CTL) contributions. The anti-FrCas^E humoral contribution is high, sustained, and principally of the IgG2a type with both in vitro neutralization- and complement-dependent cytolysis activities (23). Interestingly, it shows typical secondary response characteristics in viral challenge experiments (23), and anti-FrCas^E antibodies are transmitted transplacentally and through breastfeeding by mothers to children, where they manifest the same properties as those of 667 in the perinatal infection setting, i.e., they prevent mice from developing neurodegeneration and permit the induction of an endogenous protective antiviral immune response (25). Finally, the CTL response directed to infected cells was shown to be necessary for the protection of FrCas^E-infected, 667-treated mice, as the depletion of CD8⁺ T cells leads to death by retrovirally induced erythroleukemia (24).At this stage, an important issue is the clarification of whether the anti-FrCas^E protective immunity seen in 667 MAb-treated mice is due to actual immunomodulation by the MAb owing to its effector function(s) and/or is the consequence of viral propagation blunting, which would prevent the immune system from being overwhelmed by an excess of antigen and, hence, would give it time to react optimally. To address these two nonexclusive possibilities, we compared here viral propagations, health statuses, and endogenous immune responses in four groups of mice. The first three groups were mice neonatally infected under standard conditions (5 × 10⁴ infectious particles) and treated with either the natural 667 MAb, the antibody effector function-lacking F(ab′)₂ fragment of 667, or a neutralizing IgM (672) with effector functions inherently different from those of 667. The last group consisted of mice neonatally infected with a low FrCas^E inoculum but not subjected to immunotherapy, which is a condition permitting early viral propagation kinetics similar to those of animals infected and 667 MAb treated under standard conditions. Taken together, our data indicate that the drastic reduction of viral propagation shortly after infection is not sufficient for the induction of protective adaptive immunity and, thereby, point to an immunomodulatory action of 667. Interestingly, they also point to two sequential actions of the administered MAb. In the immediate postinfection period, viral spread is controlled exclusively by 667 neutralizing activity, and later it involves the cytolysis of infected cells owing to FcγR-binding-dependent mechanisms. Finally, our work shows that not all antibody isotypes are equally efficient at protecting infected mice and favoring the mounting of protective immunity, as 672 IgM immunotherapy-treated animals died of erythroleukemia.     

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

Background

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets.

Methodology/Principal Findings

A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested.

Conclusions/Significance

Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.     

人源抗狂犬病毒中和性全抗体在昆虫细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特异性单抗G10Fab的基因 ,克隆入杆状病毒人源IgG抗体表达载体 ,通过转染将重组质粒导入昆虫细胞 ,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体G10。用亲和层析的方法纯化了表达产物 ,经与一株鼠源糖蛋白特异性单抗竞争证实 ,该单克隆抗体特异性识别狂犬病毒糖蛋白 ,亲和力约为 10 -9M。体外中和实验证明 ,该单抗对狂犬病毒aG株具有体外中和活性     

Identification of a Critical T(Q/D/E)x5ADx2(I/L) Motif from Primate Lentivirus Vif Proteins That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x₅ADx₂(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif''s neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x₅ADx₂(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.Vif is a small viral protein that has 192 amino acids and is expressed by most lentiviruses, except for equine infectious anemia virus. It was first discovered in human immunodeficiency virus type 1 (HIV-1) (13, 14, 31), and its function in HIV-1 infection has been studied extensively (9, 34). Infection of human T-cell lines with vif-defective (ΔVif) HIV-1 identified two different cell types, namely, permissive cells that can be infected by ΔVif HIV-1 and nonpermissive cells, which are resistant to ΔVif HIV-1 (10, 36). Genomic complementation analysis indicated that these nonpermissive cells express a Vif-sensitive dominant viral inhibitor(s) (17, 27). The first inhibitor identified was APOBEC3G (A3G) (25), which belongs to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family. In humans, this family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4. All seven A3 genes have been shown to inhibit replication of various types of retrovirus by cytidine deamination-dependent and -independent mechanisms, as reviewed recently (21, 30, 35). In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit HIV-1 replication, whereas A3A and A3C do not (2, 5, 6, 25, 39, 46). Among these, the protein expression of A3G and A3F in human primary tissues has been demonstrated, and in vitro studies indicate that these proteins have the most potent anti-HIV-1 activities. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (38) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (15).Vif hijacks the cellular proteasomal machinery to destroy A3G and A3F by the protein degradation pathway (18, 26, 33, 46). Vif acts as an adaptor protein that bridges A3 proteins to a cullin 5 (Cul5)-based E3 ubiquitin ligase complex, which includes Cul5, EloB, and EloC (44). These interactions trigger the polyubiquitylation of Vif, A3G, and A3F and direct them to 26S proteasomes for degradation. Thus, Vif binding to A3G or A3F as well as to Cul5/EloBC is a critical step for A3G and A3F degradation. Although A3G and A3F share a high level of homology, different surfaces are used for Vif interaction. Vif binds to the N-terminal region of A3G, from residues 126 to 132, and to the C-terminal region of A3F, from residues 283 to 300 (12, 24). In addition, 11 discontinuous motifs in the Vif protein have been identified as regulating Vif interactions with A3G, A3F, or the Cul5/EloBC E3 ligase complex. Three motifs determine Vif interaction with the E3 ligase. The ¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹ motif, also called the HCCH zinc finger, binds to Cul5 (16, 20, 41); the ¹⁴⁴SLQYLA¹⁴⁹ motif, which is also called the BC box, binds to EloC (19, 45); and the ¹⁶¹PPLPx₄L¹⁶⁹ motif, which is also called the Cul box, binds to Cul5 (32, 45). The ¹⁶¹PPLP¹⁶⁴ subdomain has multiple activities, which not only determine Vif dimerization (43) but also regulate Vif binding to A3G (8, 37) and EloB (1). The other 8 motifs regulate the interaction between Vif and A3G/A3F. The ²¹WxSLVK²⁶ (3, 7) and ⁴⁰YRHHY⁴⁴ (23) motifs regulate Vif binding to A3G; the ¹¹Wx₂DRMR¹⁷ (23), ⁷⁴TGERxW⁷⁹ (11), and ¹⁷¹EDRW¹⁷⁴ (4) motifs regulate Vif binding to A3F; and the ⁵⁵VxIPLx₄L⁶⁴ (11), ⁶⁹YxxL⁷² (22), and ⁸¹LGxGx₂IxW⁸⁹ (4) motifs regulate Vif binding to both A3G and A3F. The ⁸¹LGxGx₂IxW⁸⁹ motif also regulates Vif binding to Cul5 (4). Thus, HIV has developed rather complicated mechanisms to assemble a protein degradation complex to neutralize these two critical host factors. A full understanding of these mechanisms is essential for pharmaceutical inhibition of Vif function to prevent HIV-1 infection. Here we report another functional motif from a previously uncharacterized region of HIV-1 Vif that regulates Vif interactions with A3G, A3F, and the Cul5/EloBC E3 ligase complex. Since this Vif region is the only one left uncharacterized, this is a significant step toward a complete understanding of this important host-pathogen interaction.     

APOBEC2 is a monomer in solution: implications for APOBEC3G models

Although the physiological role of APOBEC2 is still largely unknown, a crystal structure of a truncated variant of this protein was determined several years ago [Prochnow, C. (2007) Nature445, 447-451]. This APOBEC2 structure had considerable impact in the HIV field because it was considered a good model for the structure of APOBEC3G, an important HIV restriction factor that abrogates HIV infectivity in the absence of the viral accessory protein Vif. The quaternary structure and the arrangement of the monomers of APOBEC2 in the crystal were taken as being representative for APOBEC3G and exploited in explaining its enzymatic and anti-HIV activity. Here we show, unambiguously, that in contrast to the findings for the crystal, APOBEC2 is monomeric in solution. The nuclear magnetic resonance solution structure of full-length APOBEC2 reveals that the N-terminal tail that was removed for crystallization resides close to strand β2, the dimer interface in the crystal structure, and shields this region of the protein from engaging in intermolecular contacts. In addition, the presence of the N-terminal region drastically alters the aggregation propensity of APOBEC2, rendering the full-length protein highly soluble and not prone to precipitation. In summary, our results cast doubt on all previous structure-function predictions for APOBEC3G that were based on the crystal structure of APOBEC2.     

Lentiviral Vif Degrades the APOBEC3Z3/APOBEC3H Protein of Its Mammalian Host and Is Capable of Cross-Species Activity

All lentiviruses except equine infectious anemia virus (EIAV) use the small accessory protein Vif to counteract the restriction activity of the relevant APOBEC3 (A3) proteins of their host species. Prior studies have suggested that the Vif-A3 interaction is species specific. Here, using the APOBEC3H (Z3)-type proteins from five distinct mammals, we report that this is generally not the case: some lentiviral Vif proteins are capable of triggering the degradation of both the A3Z3-type protein of their normal host species and those of several other mammals. For instance, SIV_mac Vif can mediate the degradation of the human, macaque, and cow A3Z3-type proteins but not of the sheep or cat A3Z3-type proteins. Maedi-visna virus (MVV) Vif is similarly promiscuous, degrading not only sheep A3Z3 but also the A3Z3-type proteins of humans, macaques, cows, and cats. In contrast to the neutralization capacity of these Vif proteins, human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV) Vif appear specific to the A3Z3-type protein of their hosts. We conclude, first, that the Vif-A3Z3 interaction can be promiscuous and, second, despite this tendency, that each lentiviral Vif protein is optimized to degrade the A3Z3 protein of its mammalian host. Our results thereby suggest that the Vif-A3Z3 interaction is relevant to lentivirus biology.Lentiviruses are a unique class of complex retroviruses that encode a variety of accessory proteins in addition to the required Gag, Pol, and Env proteins. The archetypal lentivirus, human immunodeficiency virus type 1 (HIV-1), infects humans, but other members include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), maedi-visna virus (MVV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and feline immunodeficiency virus (FIV), which infect monkeys, cattle, sheep, goats, horses, and cats, respectively. The HIV-1 accessory protein viral infectivity factor (Vif) has been extensively studied because of its essential function in inhibiting the cellular antiretroviral human APOBEC3G (A3G) protein (43). HIV-1 Vif binds to human A3G (and other A3 proteins) and serves as an adaptor to link it to an ELOC-based E3 ubiquitin ligase complex (30, 51, 52). A3G is then polyubiquitinated and degraded by the cellular proteasome (7, 15, 29, 30, 43, 46, 52).Due to the potential therapeutic value of disrupting this host-pathogen interaction, a significant amount of work has been invested in defining the important contact residues between A3G and HIV-1 Vif. Primate A3G homologs have been useful tools in this effort, as many fail to be neutralized by HIV-1 Vif despite a relatively high degree of sequence similarity. For example, while HIV-1 Vif effectively neutralizes human A3G, it does not neutralize African green monkey A3G or rhesus macaque A3G despite 77% and 75% identity, respectively (4, 26, 27, 41, 51). The differential capacity of the HIV-1 and SIV_agm Vif proteins to degrade the A3G proteins of their hosts led to demonstrations that residue 128 is a key determinant: D128 made each A3G protein susceptible to HIV-1 Vif and K128 made each A3G protein susceptible to SIV_agm Vif (4, 26, 41, 51). This apparent on/off switch led to the prevailing model that the Vif-A3 interaction is species specific. However, even early data sets showed at least two hints that the story was more complex. First, the identity of the A3G residue 128 (K or D) does not diminish the interaction with the Vif proteins of SIV_mac or HIV-2 (41, 51). Second, SIV_mac Vif was shown to potently counteract the A3G proteins from rhesus macaque (as expected) but also those from human, African green monkey, and chimpanzee (27). Therefore, the implication from these studies is that the full nature of the A3-Vif interaction has yet to be elucidated.Although A3G has clearly served as the prototype for understanding the A3-Vif interaction, a growing number of studies indicate that other A3s are also capable of restricting lentivirus replication and interacting with Vif. A3G is one of seven human A3 proteins (A3A to -H) encoded in tandem on chromosome 22 (7, 16, 49). All but A3A have been implicated in the restriction of HIV-1 replication (reviewed in references 1, 10, and 45). For instance, human A3H has been shown to restrict HIV-1 replication and is susceptible to degradation by HIV-1 Vif (8, 37, 47). A3H is a Z3-type DNA deaminase characterized by a conserved threonine and a valine, in addition to the canonical H-x₁-E-x_23-28-C-x_2-4-C zinc-coordinating motif (23). The Z3-type deaminase is unique in that only one copy exists in all mammals whose genomes have been sequenced. It is encoded by a five-exon gene located at the distal end of each mammal''s A3 locus (adjacent to CBX7). Additional observations suggest that the Z3-type deaminases appear to have the capacity to restrict the Vif-deficient lentiviruses of their hosts. For example, African green monkey A3H can restrict the replication of SIV_agm and is susceptible to degradation by SIV_agm Vif, and the cat A3Z3 can restrict the replication of FIV and is susceptible to degradation by FIV Vif (33, 37, 48).Here, we take advantage of the fact that all sequenced mammals have a single A3Z3-type protein to test the hypothesis that these proteins are of general relevance to lentivirus restriction and to clarify the species-specific nature of the mammalian A3Z3/lentiviral Vif relationship. First, we ask if human, rhesus macaque, cow, sheep, and cat A3Z3-type proteins are all capable of retrovirus restriction. Second, we ask whether they are susceptible to Vif-mediated degradation in a host-specific manner. We show that each lentiviral Vif protein can indeed neutralize the Z3-type A3 protein of its host species. However, we were surprised to find that several of the Vif proteins, particularly SIV_mac and MVV Vif, can neutralize a broad number of A3Z3 proteins irrespective of the species of origin and overall degree of similarity. These data indicate that the A3-Vif interaction is more promiscuous than previously appreciated. Such broad functional flexibility may be relevant to understanding past retroviral zoonoses and predicting potential future events. We conclude that the A3Z3-Vif interaction is conserved on a macroscopic level, consistent with an important role in viral replication and particularly in species like artiodactyls and felines with fewer A3 proteins.     

Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, or A3G) and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F) are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus (HIV)-1 requires suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5, ElonginB, and ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. Domains in Vif that mediate APOBEC3 recognition have not been fully characterized. In the present study, we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif, which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion, and functional suppression of the A3G antiviral activity. Amino acids 52 to 72 of HIV-1 Vif (including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G, and this interaction was abolished by mutations of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G, yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G, suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44, which is only important for A3G suppression, the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression. On the other hand, another motif, TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition. Both the VxIPLx_4-5LxΦx₂YWxL and TGERxW motifs are highly conserved among HIV-1, HIV-2, and various simian immunodeficiency virus Vif proteins. Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3 proteins through conserved structural features that represent attractive targets for the development of novel inhibitors.     

The Roles of APOBEC3G Complexes in the Incorporation of APOBEC3G into HIV-1

Background

The incorporation of human APOBEC3G (hA3G) into HIV is required for exerting its antiviral activity, therefore the mechanism underlying hA3G virion encapsidation has been investigated extensively. hA3G was shown to form low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. The function of different forms of hA3G in its viral incorporation remains unclear.

Methodology/Principal Findings

In this study, we investigated the subcellular distribution and lipid raft association of hA3G using subcellular fractionation, membrane floatation assay and pulse-chase radiolabeling experiments respectively, and studied the correlation between the ability of hA3G to form the different complex and its viral incorporation. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.

Conclusions/Significance

Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.     

Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.