Bärbel Glass

Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.

Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy.

Switching on HIV Newly assembled human immunodeficiency virus (HIV) virions bud from the host cell as immature particles. Proteolysis of the Gag protein, which forms a structural lattice below the viral membrane, leads to the formation of mature infectious HIV. Fusion of mature HIV virions with a target cell is mediated by viral envelope (Env) proteins that occur in trimeric “spikes” on the surface of the virion. Chojnacki et al. (p. 524) used subdiffraction microscopy to show that the spikes were dispersed on the immature virion but clustered into a single focus on the mature virion. The clustering was important for infectivity. Coupling Gag proteolysis with clustering may ensure that only particles whose interior has switched to the entry mode are competent for membrane fusion. Rearrangements of the interior structural lattice cluster a surface glycoprotein as the virus preps for cell entry. Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

Background HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. Methods and Findings We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. Conclusions HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

The novel dendritic cell receptor Siglec-1 binds sialyllactose moieties on HIV-1 membrane gangliosides, thereby enhancing HIV-1 transinfection.

Gag Mutations Strongly Contribute to HIV-1 Resistance to Protease Inhibitors in Highly Drug-Experienced Patients besides Compensating for Fitness Loss

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

Structural Analysis of HIV-1 Maturation Using Cryo-Electron Tomography

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.

HIV-1 Gag Processing Intermediates Trans-dominantly Interfere with HIV-1 Infectivity

Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations. To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BackgroundThe Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition.ResultsQuantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nefs effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells.ConclusionNef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines.

Human immunodeficiency virus type 1 (HIV‐1) is a retrovirus that obtains its lipid envelope by budding through the plasma membrane of infected host cells. Various studies indicated that the HIV‐1 membrane differs from the producer cell plasma membrane suggesting virus budding from pre‐existing subdomains or virus‐mediated induction of a specialized budding membrane. To perform a comparative lipidomics analysis by quantitative mass spectrometry, we first evaluated two independent methods to isolate the cellular plasma membrane. Subsequent lipid analysis of plasma membranes and HIV‐1 purified from two different cell lines revealed a significantly different lipid composition of the viral membrane compared with the host cell plasma membrane, independent of the cell type investigated. Virus particles were significantly enriched in phosphatidylserine, sphingomyelin, hexosylceramide and saturated phosphatidylcholine species when compared with the host cell plasma membrane of the producer cells; they showed reduced levels of unsaturated phosphatidylcholine species, phosphatidylethanolamine and phosphatidylinositol. Cell type‐specific differences in the lipid composition of HIV‐1 and donor plasmamembranes were observed for plasmalogen–phosphatidylethanolamine and phosphatidylglycerol, which were strongly enriched only in HIV‐1 derived from MT‐4 cells. MT‐4 cell‐derived HIV‐1 also contained dihydrosphingomyelin as reported previously, but this lipid class was also enriched in the host cell membrane. Taken together, these data strongly support the hypothesis that HIV‐1 selects a specific lipid environment for its morphogenesis.

Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules. DOI: http://dx.doi.org/10.7554/eLife.04114.001