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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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Impact of BI-D on HIV-1 replication and production.A. SupT1 cells were infected with HIV-1 LAI and cultured in the absence or presence of BI-D (700 nM, 5x EC50). B. 293T cells were transfected with HIV-1 LAI plasmid, cultured with or wihout BI-D, and virus production was measured after 48 h. Mock treated cells were transfected with control plasmid pBluescript-SK+. Average values with SD are shown (N = 3). C. The virus stock produced in (B) was used for infection of SupT1 T cells. No additional BI-D was added during culturing. The CA-p24 level in the culture medium was monitored by ELISA.
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pone-0103552-g001: Impact of BI-D on HIV-1 replication and production.A. SupT1 cells were infected with HIV-1 LAI and cultured in the absence or presence of BI-D (700 nM, 5x EC50). B. 293T cells were transfected with HIV-1 LAI plasmid, cultured with or wihout BI-D, and virus production was measured after 48 h. Mock treated cells were transfected with control plasmid pBluescript-SK+. Average values with SD are shown (N = 3). C. The virus stock produced in (B) was used for infection of SupT1 T cells. No additional BI-D was added during culturing. The CA-p24 level in the culture medium was monitored by ELISA.

Mentions: To test the impact of ALLINIs on HIV-1 RNA processes during virion assembly and maturation, we choose the compound BI-D, which inhibits the HIV-1 NL4-3 strain on SupT1 [16] and C8166 T cells [54]. To confirm the inhibitory effect, we infected SupT1 T cells with the HIV-1 LAI strain and cultured the cells with or without BI-D (700 nM, 5x EC50). Viral spread was monitored by measuring the CA-p24 level in the culture supernatant. Whereas efficient virus replication resulting in a rapid increase in CA-p24 level was scored in the control culture, HIV-1 LAI was efficiently blocked by BI-D (Fig 1A).


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)

Impact of BI-D on HIV-1 replication and production.A. SupT1 cells were infected with HIV-1 LAI and cultured in the absence or presence of BI-D (700 nM, 5x EC50). B. 293T cells were transfected with HIV-1 LAI plasmid, cultured with or wihout BI-D, and virus production was measured after 48 h. Mock treated cells were transfected with control plasmid pBluescript-SK+. Average values with SD are shown (N = 3). C. The virus stock produced in (B) was used for infection of SupT1 T cells. No additional BI-D was added during culturing. The CA-p24 level in the culture medium was monitored by ELISA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103552-g001: Impact of BI-D on HIV-1 replication and production.A. SupT1 cells were infected with HIV-1 LAI and cultured in the absence or presence of BI-D (700 nM, 5x EC50). B. 293T cells were transfected with HIV-1 LAI plasmid, cultured with or wihout BI-D, and virus production was measured after 48 h. Mock treated cells were transfected with control plasmid pBluescript-SK+. Average values with SD are shown (N = 3). C. The virus stock produced in (B) was used for infection of SupT1 T cells. No additional BI-D was added during culturing. The CA-p24 level in the culture medium was monitored by ELISA.
Mentions: To test the impact of ALLINIs on HIV-1 RNA processes during virion assembly and maturation, we choose the compound BI-D, which inhibits the HIV-1 NL4-3 strain on SupT1 [16] and C8166 T cells [54]. To confirm the inhibitory effect, we infected SupT1 T cells with the HIV-1 LAI strain and cultured the cells with or without BI-D (700 nM, 5x EC50). Viral spread was monitored by measuring the CA-p24 level in the culture supernatant. Whereas efficient virus replication resulting in a rapid increase in CA-p24 level was scored in the control culture, HIV-1 LAI was efficiently blocked by BI-D (Fig 1A).

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