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A unique spumavirus Gag N-terminal domain with functional properties of orthoretroviral matrix and capsid.

Goldstone DC, Flower TG, Ball NJ, Sanz-Ramos M, Yap MW, Ogrodowicz RW, Stanke N, Reh J, Lindemann D, Stoye JP, Taylor IA - PLoS Pathog. (2013)

Bottom Line: Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission.Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV.These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, the Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

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Infectivity and particle budding of Gag mutants.293T cells were co-transfected with equal amounts of PFV transfer vector puc2MD9, Env packaging construct pcoPE, Pol packaging construct pcoPP and various Gag packaging constructs (wt: pcziPG CLHH; V14S: pcziPG CLHH V14S; L17S: pcziPG CLHH L17S; L21S: pcziPG CLHH L21S; L17, 21S: pcziGag4 L17, 21S) or only with pUC19 (mock) as indicated. Western blot analysis of cell lysates (cell) and pelleted viral supernatants (virus) using (A) polyclonal antibodies specific for PFV-Gag (α-Gag) or (B) rabbit polyclonal antibodies specific for PFV Env-LP (α-LP). The identity of the individual proteins is indicated on the right. (C) Relative amounts of released Gag and RT quantified from Western blots from two independent experiments (n = 2–4). (D) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay were determined 3 days post infection. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these plain supernatants were 8.7±3.3×106 EGFP ffu/ml. Means and standard deviations of three independent experiments (n = 3–6) are shown.
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ppat-1003376-g006: Infectivity and particle budding of Gag mutants.293T cells were co-transfected with equal amounts of PFV transfer vector puc2MD9, Env packaging construct pcoPE, Pol packaging construct pcoPP and various Gag packaging constructs (wt: pcziPG CLHH; V14S: pcziPG CLHH V14S; L17S: pcziPG CLHH L17S; L21S: pcziPG CLHH L21S; L17, 21S: pcziGag4 L17, 21S) or only with pUC19 (mock) as indicated. Western blot analysis of cell lysates (cell) and pelleted viral supernatants (virus) using (A) polyclonal antibodies specific for PFV-Gag (α-Gag) or (B) rabbit polyclonal antibodies specific for PFV Env-LP (α-LP). The identity of the individual proteins is indicated on the right. (C) Relative amounts of released Gag and RT quantified from Western blots from two independent experiments (n = 2–4). (D) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay were determined 3 days post infection. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these plain supernatants were 8.7±3.3×106 EGFP ffu/ml. Means and standard deviations of three independent experiments (n = 3–6) are shown.

Mentions: It has been shown previously that mutation of Leu17 in PFV-Gag-NtD gives rise to viral defects and negatively affects viral egress. Substitution by alanine has only minor effects on Env incorporation and particle release but progeny particles show a severe reduction of the infectivity. In contrast, serine substitution results in a loss of viral budding capacity [53]. To assess in vivo effects of serine substitution at Leu17 and at other positions in the Gag-Env interface the Leu17, Val14 and Leu21 to Ser mutations that disrupt the Gag-Env interaction in vitro were introduced and transfected cells assayed for particle production as well as Env/Gag incorporation and viral infectivity, Figure 6. In these in vivo experiments, the greatest effects were seen with Leu17 and Leu17/Leu21 mutant viruses that show greatly reduced levels of Gag released into the supernatant compared to wild type. By contrast, only a small reduction in Gag release was observed in the Val14 virus and in the Leu21 virus the amount of Gag is comparable to wt, Figure 6A. Examination of Env production and processing in the producer cells reveals it is unaffected by any of the mutations, Figure 6B. However, Env incorporation into virions is greatly reduced in both the Leu17 and Leu17/Leu21 particles, moderately reduced in the Val14 virus and that near wt levels are present in Leu21 particles. These results are mirrored when particle release was quantified, Figure 6C, where Leu21 particle production is only slightly reduced, Val14 is reduced around 3-fold, Leu17 around 20-fold and in the double substitution no particles are detectable in the cell supernatant. Where particles were released they were tested for infectivity relative to wt, Figure 6D. Although the Val14 mutant showed greater defects in viral Env and Gag incorporation the viruses were only around 6-fold reduced in infectivity whereas viruses with the Leu21 substitution showed around a 300 fold reduction in infectivity and no infectivity (>100,00-fold reduced) was detectable for the Leu17 mutant. Taking these data together it is apparent that the Leu17 mutation has the least effect on in vitro Env binding but causes very large defects in PFV virion production with little, if any, incorporation of viral proteins into particles. The Leu21 substitution weakens the in vitro Gag-Env interaction more, has little effect on particle production but the resulting viruses are poorly infectious and viruses with the Val14 substitution display intermediate effects having both reduced particle production and reduced infectivity.


A unique spumavirus Gag N-terminal domain with functional properties of orthoretroviral matrix and capsid.

Goldstone DC, Flower TG, Ball NJ, Sanz-Ramos M, Yap MW, Ogrodowicz RW, Stanke N, Reh J, Lindemann D, Stoye JP, Taylor IA - PLoS Pathog. (2013)

Infectivity and particle budding of Gag mutants.293T cells were co-transfected with equal amounts of PFV transfer vector puc2MD9, Env packaging construct pcoPE, Pol packaging construct pcoPP and various Gag packaging constructs (wt: pcziPG CLHH; V14S: pcziPG CLHH V14S; L17S: pcziPG CLHH L17S; L21S: pcziPG CLHH L21S; L17, 21S: pcziGag4 L17, 21S) or only with pUC19 (mock) as indicated. Western blot analysis of cell lysates (cell) and pelleted viral supernatants (virus) using (A) polyclonal antibodies specific for PFV-Gag (α-Gag) or (B) rabbit polyclonal antibodies specific for PFV Env-LP (α-LP). The identity of the individual proteins is indicated on the right. (C) Relative amounts of released Gag and RT quantified from Western blots from two independent experiments (n = 2–4). (D) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay were determined 3 days post infection. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these plain supernatants were 8.7±3.3×106 EGFP ffu/ml. Means and standard deviations of three independent experiments (n = 3–6) are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649970&req=5

ppat-1003376-g006: Infectivity and particle budding of Gag mutants.293T cells were co-transfected with equal amounts of PFV transfer vector puc2MD9, Env packaging construct pcoPE, Pol packaging construct pcoPP and various Gag packaging constructs (wt: pcziPG CLHH; V14S: pcziPG CLHH V14S; L17S: pcziPG CLHH L17S; L21S: pcziPG CLHH L21S; L17, 21S: pcziGag4 L17, 21S) or only with pUC19 (mock) as indicated. Western blot analysis of cell lysates (cell) and pelleted viral supernatants (virus) using (A) polyclonal antibodies specific for PFV-Gag (α-Gag) or (B) rabbit polyclonal antibodies specific for PFV Env-LP (α-LP). The identity of the individual proteins is indicated on the right. (C) Relative amounts of released Gag and RT quantified from Western blots from two independent experiments (n = 2–4). (D) Relative infectivity of extracellular 293T cell culture supernatants using an eGFP marker gene transfer assay were determined 3 days post infection. The values obtained using the wild type Gag packaging vector were arbitrarily set to 100%. Absolute titres of these plain supernatants were 8.7±3.3×106 EGFP ffu/ml. Means and standard deviations of three independent experiments (n = 3–6) are shown.
Mentions: It has been shown previously that mutation of Leu17 in PFV-Gag-NtD gives rise to viral defects and negatively affects viral egress. Substitution by alanine has only minor effects on Env incorporation and particle release but progeny particles show a severe reduction of the infectivity. In contrast, serine substitution results in a loss of viral budding capacity [53]. To assess in vivo effects of serine substitution at Leu17 and at other positions in the Gag-Env interface the Leu17, Val14 and Leu21 to Ser mutations that disrupt the Gag-Env interaction in vitro were introduced and transfected cells assayed for particle production as well as Env/Gag incorporation and viral infectivity, Figure 6. In these in vivo experiments, the greatest effects were seen with Leu17 and Leu17/Leu21 mutant viruses that show greatly reduced levels of Gag released into the supernatant compared to wild type. By contrast, only a small reduction in Gag release was observed in the Val14 virus and in the Leu21 virus the amount of Gag is comparable to wt, Figure 6A. Examination of Env production and processing in the producer cells reveals it is unaffected by any of the mutations, Figure 6B. However, Env incorporation into virions is greatly reduced in both the Leu17 and Leu17/Leu21 particles, moderately reduced in the Val14 virus and that near wt levels are present in Leu21 particles. These results are mirrored when particle release was quantified, Figure 6C, where Leu21 particle production is only slightly reduced, Val14 is reduced around 3-fold, Leu17 around 20-fold and in the double substitution no particles are detectable in the cell supernatant. Where particles were released they were tested for infectivity relative to wt, Figure 6D. Although the Val14 mutant showed greater defects in viral Env and Gag incorporation the viruses were only around 6-fold reduced in infectivity whereas viruses with the Leu21 substitution showed around a 300 fold reduction in infectivity and no infectivity (>100,00-fold reduced) was detectable for the Leu17 mutant. Taking these data together it is apparent that the Leu17 mutation has the least effect on in vitro Env binding but causes very large defects in PFV virion production with little, if any, incorporation of viral proteins into particles. The Leu21 substitution weakens the in vitro Gag-Env interaction more, has little effect on particle production but the resulting viruses are poorly infectious and viruses with the Val14 substitution display intermediate effects having both reduced particle production and reduced infectivity.

Bottom Line: Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission.Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV.These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, MRC National Institute for Medical Research, the Ridgeway, Mill Hill, London, United Kingdom.

ABSTRACT
The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.

Show MeSH
Related in: MedlinePlus