Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases

Herschhorn, Alon; Oz-Gleenberg, Iris; Hizi, Amnon

doi:10.1042/bj20071279

Cited by 25 publications

(22 citation statements)

References 47 publications

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“…Kinetic analysis was performed on a Biacore 3000 optical biosensor (General Electric), as previously described (52), with the following modifications. sCD4 was immobilized onto flow cells 2, 3, and 4 on a CM5 sensor chip to surface densities of ϳ700, ϳ1,400, and ϳ2,100 response units, respectively, Flow cell 1 was activated and deactivated and used as a control for nonspecific binding and refractive index changes.…”

Section: Methodsmentioning

confidence: 99%

Loss of a Conserved N-Linked Glycosylation Site in the Simian Immunodeficiency Virus Envelope Glycoprotein V2 Region Enhances Macrophage Tropism by Increasing CD4-Independent Cell-to-Cell Transmission

Yen

Herschhorn

Haim

et al. 2014

J Virol

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Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains differ in their capacity to replicate in macrophages, but mechanisms underlying these differences are not fully understood. Here, we identify a highly conserved Nlinked glycosylation site (N173 in SIV, corresponding to N160 in HIV) in the V2 region of the SIV envelope glycoprotein (Env) as a novel determinant of macrophage tropism and characterize mechanisms underlying this phenotype. Loss of the N173 glycosylation site in the non-macrophage-tropic SIVmac239 by introducing an N173Q mutation enhanced viral replication and multinucleated giant cell formation upon infection of rhesus macrophages, while the addition of N173 to SIVmac251 had the opposite effect. The removal of N173 in SIVmac239 enhanced CD4-independent cell-to-cell transmission to CCR5-expressing cells. SIVmac239 with N173Q mediated CD4-independent cell-cell fusion but could not infect CD4-negative cells in single-round infections. Thus, CD4-independent phenotypes were detected only in the context of cell-to-cell contact. Similar results were obtained in SIVmac251 with and without N173. N173 decreased the neutralization sensitivity of SIVmac251 but had no effect on the neutralization sensitivity of SIVmac239. The N173Q mutation had no effect on SIVmac239 binding to CD4 in Biacore assays, coimmunoprecipitation assays, and enzyme-linked immunosorbent assays (ELISAs). These findings suggest that the loss of the N173 N-linked glycosylation site increases SIVmac239 replication in macrophages by enhancing CD4-independent cell-to-cell virus transmission through CCR5-mediated fusion. This mechanism may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses in vivo. IMPORTANCEIn this study, we identify a genetic determinant in the viral envelope (N173) that increases replication and spreading infection of SIV strains in macrophages by enhancing cell-to-cell virus transmission. This effect is explained by a novel mechanism involving increased cell-to-cell fusion in the absence of CD4, the primary receptor that normally mediates virus entry. The same genetic determinant also affects the sensitivity of these viruses to inhibition by neutralizing antibodies. Most macrophage-tropic HIV/ SIV strains are known to be neutralization sensitive. Together, these findings suggest that this efficient mode of virus transmission may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses to cells expressing little or no CD4 in vivo. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains differ in their ability to infect and replicate in macrophages. Macrophage tropism is determined primarily by the envelope glycoprotein (Env), which forms trimers composed of three noncovalently bound heterodimers of gp120 and gp41 subunits. The gp120 exterior subunit interacts with CD4 and a coreceptor, mainly CCR5 (1, 2),...

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Section: Methodsmentioning

confidence: 99%

Loss of a Conserved N-Linked Glycosylation Site in the Simian Immunodeficiency Virus Envelope Glycoprotein V2 Region Enhances Macrophage Tropism by Increasing CD4-Independent Cell-to-Cell Transmission

Yen

Herschhorn

Haim

et al. 2014

J Virol

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“…Integrase binds the HIV-1 RT heterodimer (p66/ p51); conversely, the individual RT subunits, p51 and p66, are each able to bind IN (7). These interactions appear to promote viral replication, although some studies show that RT inhibits the enzymatic activities of IN (8,9).…”

mentioning

confidence: 99%

Altered Viral Fitness and Drug Susceptibility in HIV-1 Carrying Mutations That Confer Resistance to Nonnucleoside Reverse Transcriptase and Integrase Strand Transfer Inhibitors

Kuritzkes

2014

J Virol

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Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) and integrase (IN) strand transfer inhibitors (INSTI)are key components of antiretroviral regimens. To explore potential interactions between NNRTI and INSTI resistance mutations, we investigated the combined effects of these mutations on drug susceptibility and fitness of human immunodeficiency virus type 1 (HIV-1). In the absence of drug, single-mutant viruses were less fit than the wild type; viruses carrying multiple mutations were less fit than single-mutant viruses. These findings were explained in part by the observation that mutant viruses carrying NNRTI plus INSTI resistance mutations had reduced amounts of virion-associated RT and/or IN protein. In the presence of efavirenz (EFV), a virus carrying RT-K103N together with IN-G140S and IN-Q148H (here termed IN-G140S/Q148H) mutations was fitter than a virus with a RT-K103N mutation alone. Similarly, in the presence of EFV, the RT-E138K plus IN-G140S/Q148H mutant virus was fitter than one with the RT-E138K mutation alone. No effect of INSTI resistance mutations on the fitness of RT-Y181C mutant viruses was observed. Conversely, RT-E138K and -Y181C mutations improved the fitness of the IN-G140S/Q148H mutant virus in the presence of raltegravir (RAL); the RT-K103N mutation had no effect. The NNRTI resistance mutations had no effect on RAL susceptibility. Likewise, the IN-G140S/Q148H mutations had no effect on EFV or RPV susceptibility. However, both the RT-K103N plus IN-G140S/Q148H and the RT-E138K plus IN-G140S/Q148H mutant viruses had significantly greater fold increases in 50% inhibitory concentration (IC 50 ) of EFV than viruses carrying a single NNRTI mutation. Likewise, the RT-E138K plus IN-G140S/Q148H mutant virus had significantly greater fold increases in RAL IC 50 than that of the IN-G140S/ Q148H mutant virus. These results suggest that interactions between RT and IN mutations are important for NNRTI and INSTI resistance and viral fitness. IMPORTANCENonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit.

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“…HIV-1 RT has been identified to physically interact with viral integrase by using GST pulldown assays, coimmunoprecipitation assays, dot blot assays, NMR spectroscopy analyses, and surface plasmon resonance analyses (101)(102)(103)(104)(105)(106)(107)(108). The binding of integrase to RT does not require multimeric integrase or an integrase with complete enzymatic activity (108).…”

Section: Rt-integrase Interactionmentioning

confidence: 99%

“…HIV integrase has been determined to physically interact with RT by using GST pulldown assays, coimmunoprecipitation assays, dot blot assays, NMR spectroscopy, and surface plasmon resonance analyses (101)(102)(103)(104)(105)(106)(107). Two functions of the integrase-RT interaction have been proposed.…”

Section: Integrase-rt Interactionmentioning

confidence: 99%

“…Two functions of the integrase-RT interaction have been proposed. First, RT in the PIC inhibits both the 3=-end endonuclease and the strand transfer activity of integrase (101,103). Second, RT can inhibit the DNA disintegration activity of integrase before viral integration, although HIV-1 integrase may promote RT activity during reverse transcription (171).…”

Section: Integrase-rt Interactionmentioning

confidence: 99%

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HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Clercq

2016

Microbiol Mol Biol Rev

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SUMMARYThe HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle.

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Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases

Cited by 25 publications

References 47 publications

Loss of a Conserved N-Linked Glycosylation Site in the Simian Immunodeficiency Virus Envelope Glycoprotein V2 Region Enhances Macrophage Tropism by Increasing CD4-Independent Cell-to-Cell Transmission

Loss of a Conserved N-Linked Glycosylation Site in the Simian Immunodeficiency Virus Envelope Glycoprotein V2 Region Enhances Macrophage Tropism by Increasing CD4-Independent Cell-to-Cell Transmission

Altered Viral Fitness and Drug Susceptibility in HIV-1 Carrying Mutations That Confer Resistance to Nonnucleoside Reverse Transcriptase and Integrase Strand Transfer Inhibitors

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

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