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The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

van Bel N, van der Velden Y, Bonnard D, Le Rouzic E, Das AT, Benarous R, Berkhout B - PLoS ONE (2014)

Bottom Line: It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes.The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo.In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

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Activity of virus-extracted RT enzyme.Virus was produced with or without BI-D. Virus supernatant was incubated with an MS2 RNA template and dNTPs to reverse transcribe the template RNA. The activity of the RT enzyme was determined by quantitation of the cDNA product by qPCR. Serial dilutions of AMV-RT were used to generate a standard curve. Average values with SD are shown (N = 2). Mock: supernatant of cells transfected with control plasmid, pBluescript-SK+.
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pone-0103552-g004: Activity of virus-extracted RT enzyme.Virus was produced with or without BI-D. Virus supernatant was incubated with an MS2 RNA template and dNTPs to reverse transcribe the template RNA. The activity of the RT enzyme was determined by quantitation of the cDNA product by qPCR. Serial dilutions of AMV-RT were used to generate a standard curve. Average values with SD are shown (N = 2). Mock: supernatant of cells transfected with control plasmid, pBluescript-SK+.

Mentions: Virus was produced by 293T cells in the presence or absence of BI-D, yielding similar CA-p24 levels. The RT enzyme was extracted from virions in the culture supernatant by addition of Triton-X100 and used to reverse transcribe an MS2 RNA template with an annealed DNA primer upon addition of dNTPs. The cDNA product was quantified by real-time PCR (qPCR) using a MS2-specific probe. After correction for the virus input based on CA-p24, we measured a similar RT activity in the extracts of viruses generated with or without BI-D (Fig 4). This result indicates that BI-D does not affect RT production and activity.


The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

van Bel N, van der Velden Y, Bonnard D, Le Rouzic E, Das AT, Benarous R, Berkhout B - PLoS ONE (2014)

Activity of virus-extracted RT enzyme.Virus was produced with or without BI-D. Virus supernatant was incubated with an MS2 RNA template and dNTPs to reverse transcribe the template RNA. The activity of the RT enzyme was determined by quantitation of the cDNA product by qPCR. Serial dilutions of AMV-RT were used to generate a standard curve. Average values with SD are shown (N = 2). Mock: supernatant of cells transfected with control plasmid, pBluescript-SK+.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114784&req=5

pone-0103552-g004: Activity of virus-extracted RT enzyme.Virus was produced with or without BI-D. Virus supernatant was incubated with an MS2 RNA template and dNTPs to reverse transcribe the template RNA. The activity of the RT enzyme was determined by quantitation of the cDNA product by qPCR. Serial dilutions of AMV-RT were used to generate a standard curve. Average values with SD are shown (N = 2). Mock: supernatant of cells transfected with control plasmid, pBluescript-SK+.
Mentions: Virus was produced by 293T cells in the presence or absence of BI-D, yielding similar CA-p24 levels. The RT enzyme was extracted from virions in the culture supernatant by addition of Triton-X100 and used to reverse transcribe an MS2 RNA template with an annealed DNA primer upon addition of dNTPs. The cDNA product was quantified by real-time PCR (qPCR) using a MS2-specific probe. After correction for the virus input based on CA-p24, we measured a similar RT activity in the extracts of viruses generated with or without BI-D (Fig 4). This result indicates that BI-D does not affect RT production and activity.

Bottom Line: It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes.The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo.In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Show MeSH
Related in: MedlinePlus