Modulation of human immunodeficiency virus type 1 infectivity through incorporation of tetraspanin proteins

Accumulating evidence indicates that human immunodeficiency virus type 1 (HIV-1) acquires various cellular membrane proteins in the lipid bilayer of the viral envelope membrane. Although some virion-incorporated cellular membrane proteins are known to potently affect HIV-1 infectivity, the virological functions of most virion-incorporated membrane proteins remain unclear. Among these host proteins, we found that CD63 was eliminated from the plasma membranes of HIV-1-producing T cells after activation, followed by a decrease in the amount of virion-incorporated CD63, and in contrast, an increase in the infectivity of the released virions. On the other hand, we found that CD63 at the cell surface was preferentially embedded on the membrane of released virions in an HIV-1 envelope protein (Env)-independent manner and that virion-incorporated CD63 had the potential to inhibit HIV-1 Env-mediated infection in a strain-specific manner at the postattachment entry step(s). In addition, these behaviors were commonly observed in other tetraspanin proteins, such as CD9, CD81, CD82, and CD231. However, L6 protein, whose topology is similar to that of tetraspanins but which does not belong to the tetraspanin superfamily, did not have the potential to prevent HIV-1 infection, despite its successful incorporation into the released particles. Taken together, these results suggest that tetraspanin proteins have the unique potential to modulate HIV-1 infectivity through incorporation into released HIV-1 particles, and our findings may provide a clue to undiscovered aspects of HIV-1 entry.     

Redox-triggered infection by disulfide-shackled human immunodeficiency virus type 1 pseudovirions

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant's infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.     

Importin 7 may be dispensable for human immunodeficiency virus type 1 and simian immunodeficiency virus infection of primary macrophages

In an in vitro assay employing reconstituted nuclei, importin 7 (IPO7) has been implicated in nuclear translocation of human immunodeficiency virus type 1 (HIV-1) cDNA. Using RNA interference technology, we inhibited expression of IPO7 by 80 to 95% in primary macrophages and in HeLa cells and monitored their ability to support HIV-1 and simian immunodeficiency virus (SIV) cDNA synthesis, nuclear translocation, and infection efficiency. Marked IPO7 deficiency did not alter the rate or extent of HIV-1 or SIV cDNA synthesis or nuclear translocation. The infection efficiency of HIV-1 was similarly unaltered. Therefore, in natural, nondividing targets of HIV-1, IPO7 may be dispensable for infection.     

Multiple extracellular domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion.

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.     

Tissue-resident macrophages are productively infected ex vivo by primary X4 isolates of human immunodeficiency virus type 1

Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.     

Degradation of cytoskeletal proteins by the human immunodeficiency virus type 1 protease.

Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with M_r ∼ 90,000 and a protein with M_r ∼ 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease.     

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.     

Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.

We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.     

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.     

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.     

Modest but reproducible inhibition of human immunodeficiency virus type 1 infection in macrophages following LEDGFp75 silencing

LEDGFp75 is a cellular protein which binds human immunodeficiency virus type 1 (HIV-1) integrase with high specificity and affinity but whose function in infection has not been defined. We infected LEDGFp75-deficient primary macrophages with wild-type HIV in order to assess potential infection phenotypes which would provide clues to LEDGFp75 function. Silencing of LEDGFp75 by 70 to 80% resulted in an average of 53% reduced infection of macrophages by HIV. Analysis of infection intermediates showed that integration, but not two-long-terminal-repeat (2LTR) circles or late cDNAs, was reduced up to 74% in LEDGFp75-deficient macrophages. Therefore, LEDGFp75 has a modest involvement in HIV-1 integration in macrophages.     

Histone deacetylase 6 regulates human immunodeficiency virus type 1 infection

Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical alpha-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated alpha-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of alpha-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.