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Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

Esteva MJ, Affranchino JL, González SA - PLoS ONE (2014)

Bottom Line: Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex.Furthermore, we show here that the carboxyl-terminus domain (CTD) of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD.Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Virología, Universidad de Belgrano (UB) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

ABSTRACT
To gain insight into the functional relationship between the capsid (CA) domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD) of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD) of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

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Assembly phenotype of the chimeric SIVFIV CA virus.293T cells were transfected in parallel with the wild-type SIVSMM-PBj and SIVFIV CA proviral DNAs. At 48 h post-transfection, cell and virion lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and detected with antibodies specific for the SIV MA (A and C) and for the SIV and FIV CA proteins (B and D). The positions of the wild-type and chimeric Gag proteins as well as those of the MA and CA proteins are shown. Numbers refer to the positions of the molecular weight standards (in kDa).
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pone-0114299-g002: Assembly phenotype of the chimeric SIVFIV CA virus.293T cells were transfected in parallel with the wild-type SIVSMM-PBj and SIVFIV CA proviral DNAs. At 48 h post-transfection, cell and virion lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and detected with antibodies specific for the SIV MA (A and C) and for the SIV and FIV CA proteins (B and D). The positions of the wild-type and chimeric Gag proteins as well as those of the MA and CA proteins are shown. Numbers refer to the positions of the molecular weight standards (in kDa).

Mentions: To determine whether the FIV CA is able to functionally replace the equivalent SIV domain and confer assembly competence to the chimeric SIV Gag, we transfected the wild-type SIV and chimeric SIVFIV CA proviral DNAs into 293T cells. Analysis of the cell and virion lysates by Western blotting using an antiserum directed against the SIV MA showed that the chimeric SIVFIV CA Gag polyprotein was expressed and processed at wild-type levels and that it assembled into virions (Fig. 2A and 2C). However, probing for the SIV and FIV CA proteins revealed that the chimeric SIVFIV CA particles contained, besides the mature FIV CA protein, additional low-molecular-weight bands derived from the FIV CA (Fig. 2B and 2D), which suggests that maturation of the chimeric SIVFIV CA virions is accompanied by certain degree of instability of the FIV CA domain.


Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

Esteva MJ, Affranchino JL, González SA - PLoS ONE (2014)

Assembly phenotype of the chimeric SIVFIV CA virus.293T cells were transfected in parallel with the wild-type SIVSMM-PBj and SIVFIV CA proviral DNAs. At 48 h post-transfection, cell and virion lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and detected with antibodies specific for the SIV MA (A and C) and for the SIV and FIV CA proteins (B and D). The positions of the wild-type and chimeric Gag proteins as well as those of the MA and CA proteins are shown. Numbers refer to the positions of the molecular weight standards (in kDa).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4252113&req=5

pone-0114299-g002: Assembly phenotype of the chimeric SIVFIV CA virus.293T cells were transfected in parallel with the wild-type SIVSMM-PBj and SIVFIV CA proviral DNAs. At 48 h post-transfection, cell and virion lysates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and detected with antibodies specific for the SIV MA (A and C) and for the SIV and FIV CA proteins (B and D). The positions of the wild-type and chimeric Gag proteins as well as those of the MA and CA proteins are shown. Numbers refer to the positions of the molecular weight standards (in kDa).
Mentions: To determine whether the FIV CA is able to functionally replace the equivalent SIV domain and confer assembly competence to the chimeric SIV Gag, we transfected the wild-type SIV and chimeric SIVFIV CA proviral DNAs into 293T cells. Analysis of the cell and virion lysates by Western blotting using an antiserum directed against the SIV MA showed that the chimeric SIVFIV CA Gag polyprotein was expressed and processed at wild-type levels and that it assembled into virions (Fig. 2A and 2C). However, probing for the SIV and FIV CA proteins revealed that the chimeric SIVFIV CA particles contained, besides the mature FIV CA protein, additional low-molecular-weight bands derived from the FIV CA (Fig. 2B and 2D), which suggests that maturation of the chimeric SIVFIV CA virions is accompanied by certain degree of instability of the FIV CA domain.

Bottom Line: Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex.Furthermore, we show here that the carboxyl-terminus domain (CTD) of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD.Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Virología, Universidad de Belgrano (UB) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

ABSTRACT
To gain insight into the functional relationship between the capsid (CA) domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD) of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD) of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

Show MeSH
Related in: MedlinePlus