CD160 Plays a Protective Role During Chronic Infection by Enhancing Both Functionalities and Proliferative Capacity of CD8+ T Cells.

The understanding of protective immunity during HIV infection remains elusive. Here we showed that CD160 defines a polyfunctional and proliferative CD8+ T cell subset with a protective role during chronic HIV-1 infection. CD160+ CD8+ T cells derived from HIV+ patients correlated with slow progressions both in a cross-sectional study and in a 60-month longitudinal cohort, displaying enhanced cytotoxicity and proliferative capacity in response to HIV Gag stimulation; triggering CD160 promoted their functionalities through MEK-ERK and PI3K-AKT pathways. These observations were corroborated by studying chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The genetic ablation of CD160 severely impaired LCMV-specific CD8+ T cell functionalities and thereby resulted in loss of virus control. Interestingly, transcriptional profiling showed multiple costimulatory and survival pathways likely to be involved in CD160+ T cell development. Our data demonstrated that CD160 acts as a costimulatory molecule positively regulating CD8+ T cells during chronic viral infections, thus representing a potential target for immune intervention.

Reaction-diffusion basis of retroviral infectivity.

Retrovirus particle (virion) infectivity requires diffusion and clustering of multiple transmembrane envelope proteins (Env3) on the virion exterior, yet is triggered by protease-dependent degradation of a partially occluding, membrane-bound Gag polyprotein lattice on the virion interior. The physical mechanism underlying such coupling is unclear and only indirectly accessible via experiment. Modelling stands to provide insight but the required spatio-temporal range far exceeds current accessibility by all-atom or even coarse-grained molecular dynamics simulations. Nor do such approaches account for chemical reactions, while conversely, reaction kinetics approaches handle neither diffusion nor clustering. Here, a recently developed multiscale approach is considered that applies an ultra-coarse-graining scheme to treat entire proteins at near-single particle resolution, but which also couples chemical reactions with diffusion and interactions. A model is developed of Env3 molecules embedded in a truncated Gag lattice composed of membrane-bound matrix proteins linked to capsid subunits, with freely diffusing protease molecules. Simulations suggest that in the presence of Gag but in the absence of lateral lattice-forming interactions, Env3 diffuses comparably to Gag-absent Env3 Initial immobility of Env3 is conferred through lateral caging by matrix trimers vertically coupled to the underlying hexameric capsid layer. Gag cleavage by protease vertically decouples the matrix and capsid layers, induces both matrix and Env3 diffusion, and permits Env3 clustering. Spreading across the entire membrane surface reduces crowding, in turn, enhancing the effect and promoting infectivity.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

Cellular and viral determinants of retroviral nuclear entry.

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Viral factors involved in adapter-related protein complex 2 alpha 1 subunit-mediated regulation of human immunodeficiency virus type 1 replication.

The presence of siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) enhances human immunodeficiency virus type 1 (HIV-1) replication by up-regulating nuclear transport of viral genome. In this report, we examined possible viral factors involved in AP2alpha-mediated regulation of HIV-1 replication, namely, Gag matrix protein (MA), integrase (IN) and Vpr. Replication of mutant viruses lacking the nucleophilic property of one of these viral proteins was significantly enhanced by treating cells with AP2alpha siRNA, indicating that Gag MA, IN or Vpr is not specifically involved in AP2alpha-mediated enhancement of viral replication. In contrast, AP2alpha siRNA showed no effect on the level of gene transduction mediated by HIV-1-derived lentiviral vector (LV). Although virus-like LV particle and parental HIV-1 particle are composed of almost equivalent viral structural proteins, LV particles lack three accessory proteins, Vif, Vpr and Vpu, and a large portion of the HIV-1 genome. Vif, Vpr and Vpu were dispensable for AP2alpha siRNA-mediated enhancement of HIV-1 replication, indicating that a particular part of the HIV-1 genomic fragment deleted in the LV genome might be required for the enhancing effect of AP2alpha siRNA on viral replication. Taken together, these results suggest that an as yet undetermined gene fragment of the HIV-1 genome is involved in AP2alpha-mediated regulation of HIV-1 replication.

Correlation between CD4+ T-cell loss and Gag-specific T cells in different intestinal sites of chronically SIV-infected rhesus monkeys.

OBJECTIVE: To determine the loss of CD4+ T cells and virus-specific cytotoxic T cells (CTL) in different mucosal sites of rhesus monkeys infected with simian immunodeficiency virus (SIV). DESIGN: A cross-sectional comparative investigation of seven different mucosal sites from chronically SIV-infected rhesus monkeys was performed by analyzing blood and mucosal lymphocytes. METHODS: Mucosal lymphocytes were isolated from duodenum, jejunum, ileum and colon as well as from vagina, cervix and uterus of SIV-infected rhesus monkeys at necropsy. CD4+ T cells and SIV-Gag-specific CTL were determined in blood and mucosal samples by flow cytometry. RESULTS: A significant depletion of CD4+ T cells was observed in blood and all mucosal sites of SIV-infected rhesus monkeys compared to uninfected animals. But the mean percentage loss of CD4+ T cells varied between 66 and 95% between the different mucosal tissues. The frequency of CTL ranged between 0.4 and 2.4% with the highest proportions in vagina and cervix. Among the intestinal sites the mean levels of CTL correlated with mean percentage loss of CD4+ T cells. CONCLUSION: A discriminative pronounced loss of CD4+ T cells among the mucosal tissues confirmed that viral replication affects different mucosal sites in a distinct way. Despite high levels of CTL, especially in vagina and cervix, the severe loss of mucosal CD4+ T cells could not be prevented during chronic SIV infection. However, within the four sites of the intestine a high virus-specific cellular immune response correlated with better preservation of CD4+ T cells.

Analysis of the initiating events in HIV-1 particle assembly and genome packaging.

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome.

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

[Envelope virus assembly and budding].

For many enveloped viruses, viral matrix and retroviral Gag proteins are able to bud from the cell surface by themselves in the form of lipid-enveloped, virus-like particles (VLPs), suggesting that these proteins play important roles in viral assembly and budding. The major three-types of L-domain motifs, PPxY, P(T/S)AT, and YP(x)(n)L have been identified within these proteins. Many viruses have been shown to commonly utilize cellular ESCRT pathway via direct interaction between the L-domains and the components of the pathway for efficient viral budding. However, for many enveloped viruses, L-domain motifs have not yet been identified, and the involvement of the ESCRT pathway in virus budding is still unknown. Among such viruses, we have been focusing on Sendai virus (SeV) and shown that (i) SeV M functionally and physically interact with a component of the ESCRT complex, Alix/AIP1, although budding of M-VLPs does not seem to be dependent on the pathway; (ii) one of the accessory proteins of SeV, C, also interact with Alix/AIP1, and recruit it to the plasma membrane for efficient budding of M-VLPs; (iii) the C protein regulate balance of viral genome and antigenome RNA synthesis for optimized production of infectious virus particles. These results demonstrate a unique mechanism for budding of SeV as well as a novel mechanism of regulated synthesis of viral genome RNAs for efficient production of infectious particles.

The noncanonical Gag domains p8 and n are critical for assembly and release of mouse mammary tumor virus.

The mouse mammary tumor virus (MMTV) Gag contains the unique domains pp21, p3, p8, and n. We investigated the contribution of these domains to particle assembly and found that the region spanning the p8 and n domains is critical for shape determination and assembly. Deletion of pp21 and p3 reduced the number of released particles, but deletion of the n domain resulted in frequent formation of aberrant particles, while deletion of p8 severely impaired assembly. Further investigation of p8 revealed that both the basic and the proline-rich motifs within p8 contribute to MMTV assembly.

The glycosylated Gag protein of a murine leukemia virus inhibits the antiretroviral function of APOBEC3.

APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.

FIV Gag: virus assembly and host-cell interactions.

Infection of domestic cats with virulent strains of the feline immunodeficiency virus (FIV) leads to an acquired immunodeficiency syndrome (AIDS), similar to the pathogenesis induced in humans by infection with human immunodeficiency virus type 1 (HIV-1). Thus, FIV is a highly relevant model for anti-HIV therapy and vaccine development. FIV is not infectious in humans, so it is also a potentially effective non-toxic gene therapy vector. To make better use of this model, it is important to define the cellular machinery utilized by each virus to produce virus particles so that relevant similarities can be identified. It is well understood that all replication-competent retroviruses encode gag, pol, and env genes, which provide core elements for virus replication. As a result, most antiretroviral therapy targets pol-derived enzymes (protease, reverse transcriptase, and integrase) orenv-derived glycoproteins that mediate virus attachment and entry. However, resistance to drugs against these targets is a persistent problem, and novel targets must be identified to produce more effective drugs that can either substitute or be combined with current therapy. Elements of the gag gene (matrix, capsid, nucleocapsid, and "late" domains) have yet to be exploited as antiviral targets, even though the Gag precursor polyprotein is self-sufficient for the assembly and release of virus particles from cells. This process is far better understood in primate lentiviruses, especially HIV-1. However, there has been significant progress in recent years in defining how FIV Gag is targeted to the cellular plasma membrane, assembles into virions, incorporates FIV Env glycoproteins, and utilizes host cell machinery to complete virus release. Recent discoveries of intracellular restriction factors that target HIV-1 and FIV capsids after virus entry have also opened exciting new areas of research. This review summarizes currently known interactions involving HIV-1 and FIV Gag that affect virus release, infectivity, and replication.

Human immunodeficiency virus type-1 gag and host vesicular trafficking pathways.

The Gag protein of HIV-1 directs the particle assembly process. Gag recruits components of the cellular vesicular trafficking machinery in order to traverse the cytoplasm of the cell and reach the particle assembly site. The plasma membrane is the primary site of particle assembly in most cell types, while in macrophages an unusual intracellular membrane-bound compartment bearing markers of late endosomes and the plasma membrane is the predominant assembly site. Plasma membrane specificity of assembly may be directed by components of lipid rafts and the cytoplasmic leaflet component PI(4,5)P(2). Recent work has highlighted the role of adaptor protein complexes, protein sorting and recycling pathways, components of the multivesicular body, and cellular motor proteins in facilitating HIV assembly and budding. This review presents an overview of the relevant vesicular trafficking pathways and describes the individual components implicated in interactions with Gag.

HIV and SIV infection: the role of cellular restriction and immune responses in viral replication and pathogenesis.

The human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have a long biological history. Both viruses evolved from Africa and remnants of them can be found in the 'fossil record' of several species in which they are not endemic. SIV remains endemic in several species of monkeys in Africa where it does not cause immune deficiency. HIV and SIV actively replicate within humans and Asian non-human primates, despite cellular and genetic viral restriction factors and genes, and at times robust innate and adaptive immune responses. While Lentiviruses are considered 'slow viruses' it is clear in humans and susceptible Asian monkeys that virus production is rapid and highly active. This results in a massive loss of CD4+ memory effector T cells early after infection and a continued race between viral evolution, cytotoxic lymphocytes, and failed neutralizing antibody responses. Concurrently, HIV and SIV can infect monocyte/macrophage populations in blood and more importantly in tissues, including the central nervous system, where the virus can remain sequestered and not cleared by anti-retroviral therapy, and hide for years. This review will discuss species and cellular barriers to infection, and the role of innate and acquired immunity with infection and pathogenesis of HIV and SIV in select species.

Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag.

Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.

Amino acid alterations in Gag that confer the ability to grow in simian cells on HIV-1 are located at a narrow CA region.

We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.

A single amino acid substitution in a segment of the CA protein within Gag that has similarity to human immunodeficiency virus type 1 blocks infectivity of a human endogenous retrovirus K provirus in the human genome.

Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. However, no single HERV-K provirus in the human genome today appears to be infectious. Since the Gag protein is the central component for the production of retrovirus particles, we investigated the abilities of Gag from two HERV-K proviruses to support production of virus-like particles and viral infectivity. HERV-K113 has full-length open reading frames for all viral proteins, while HERV-K101 has a full-length gag open reading frame and is expressed in human male germ cell tumors. The Gag of HERV-K101 allowed production of viral particles and infectivity, although at lower levels than observed with a consensus sequence Gag. Thus, including HERV-K109, at least two HERV-K proviruses in human genome today have functional Gag proteins. In contrast, HERV-K113 Gag supported only very low levels of particle production, and no infectivity was detectable due to a single amino acid substitution (I516M) near the extreme C terminus of the CA protein within Gag. The sequence of this portion of HERV-K CA showed similarities to that of human immunodeficiency virus type 1 and other primate immunodeficiency viruses. The extreme C terminus of CA may be a general determinant of retrovirus particle production. In addition, precise mapping of the defects in HERV-K proviruses as was done here identifies the key polymorphisms that need to be analyzed to assess the possible existence of infectious HERV-K alleles within the human population.

Model of human immunodeficiency virus budding and self-assembly: role of the cell membrane.

Budding from the plasma membrane of the host cell is an indispensable step in the life cycle of the human immunodeficiency virus (HIV), which belongs to a large family of enveloped RNA viruses, retroviruses. Unlike regular enveloped viruses, retrovirus budding happens concurrently with the self-assembly of the main retrovirus protein subunits (called Gag protein after the name of the genetic material that codes for this protein: Group-specific AntiGen) into spherical virus capsids on the cell membrane. Led by this unique budding and assembly mechanism, we study the free energy profile of retrovirus budding, taking into account the Gag-Gag attraction energy and the membrane elastic energy. We find that if the Gag-Gag attraction is strong, budding always proceeds to completion. During early stage of budding, the zenith angle of partial budded capsids, alpha , increases with time as alpha proportional t1/2. However, if the Gag-Gag attraction is weak, a metastable state of partial budding appears. The zenith angle of these partially spherical capsids is given by alpha0 approximately (tau2/kappasigma)1/4 in a linear approximation, where kappa and sigma are the bending modulus and the surface tension of the membrane, and tau is a line tension of the capsid proportional to the strength of Gag-Gag attraction. Numerically, we find alpha0<0.3pi without any approximations. Using experimental parameters, we show that HIV budding and assembly always proceed to completion in normal biological conditions. On the other hand, by changing Gag-Gag interaction strength or membrane rigidity, it is relatively easy to tune it back and forth between complete budding and partial budding. Our model agrees reasonably well with experiments observing partial budding of retroviruses including HIV.

Mapping of nucleocapsid residues important for HIV-1 genomic RNA dimerization and packaging.

Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity, and the nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) facilitates HIV-1 gRNA dimerization. To identify the relevant and dispensable positions of NC, 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants prepared by site-directed mutagenesis. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The N-terminal and linker segments appeared to play predominantly electrostatic and steric roles, respectively. Mutating the hydrophobic patch of either zinc finger, or substituting alanines for their glycine doublet, was as disabling as deleting the corresponding finger. Replacing the CysX(2)CysX(4)HisX(4)Cys motif of either finger by CysX(2)CysX(4)CysX(4)Cys or CysX(2)CysX(4)HisX(4)His, interchanging the zinc fingers or, replacing one zinc finger by a copy of the other one, had generally intermediate effects; among these mutations, the His23-->Cys substitution in the N-terminal zinc finger had the mildest effect. The charge of NC could be increased or decreased by up to 18%, that of the linker could be reduced by 75% or increased by 50%, and one or two electric charges could be added or subtracted from either zinc finger, without affecting gRNA dimerization. Shortening, lengthening, or making hydrophobic the linker was as disabling as deleting the N-terminal or the C-terminal zinc finger, but a neutral and polar linker was innocuous. The present work multiplies by 4 and by 33 the number of retroviral and lentiviral NC mutations known to inhibit gRNA dimerization, respectively. It shows the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein. Most, but not all, mutations inhibited gRNA packaging; some had a strong effect on virus assembly or stability.

[Revisiting HIV-1 assembly]. / Une nouvelle vision de l'assemblage du VIH-1.

During the late stage of virus replication, incorporation of the envelope glycoproteins (Env) by Gag cores takes place together with the proteolytic maturation of Gag and Gag-Pol precursors. Assembly is initially driven by Gag oligomerisation, which requires two platorms. The first one is formed by specific membrane subdomains with which Gag molecules interact via the N-terminal MA domain, and the second by the viral genomic RNA undergoing specific interactions with the NC domain of Gag. To complete viral budding, the Gag "late domain" subsequently associates with members of the ESCRT complexes involved in the budding of vesicles in late endosomes (LE). While the cellular trafficking of the viral components is still poorly understood, there is an ongoing debate on the site of HIV-1 assembly, because this process might take place either at the plasma membrane or in intracellular compartments such as the LE, depending on the virus/cell system studied. This site may depend on the interplay of multiple overlapping trafficking signals bear by Gag and Env. Our recent results indicate that it may rely on the chronic or acute nature of the viral infection more than on the cell type. In chronically infected cells, virions probably assemble and accumulate in intracellular compartments hidden from the immune system. Release of virions in the form of bursts would be triggered during cell-cell interactions, through a specialized structure called the virological synapse.