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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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Sedimentation velocity analysis of Gag-Env interface mutants.C(S) functions that best fit sedimentation velocity profiles from (A) wt PFV-Gag-NtD, (B) V14S, (C) L17S, (D) V14S/L21S and (E) N29Q mutants. The C(S) function from 75 µM Gag-NtD (black) and from 75 µM equimolar mixtures of Gag-NtDs with Env(1–20) (red) are shown in each panel. (F) Histogram of equilibrium association constants derived from the sedimentation data as described in methods.
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ppat-1003376-g005: Sedimentation velocity analysis of Gag-Env interface mutants.C(S) functions that best fit sedimentation velocity profiles from (A) wt PFV-Gag-NtD, (B) V14S, (C) L17S, (D) V14S/L21S and (E) N29Q mutants. The C(S) function from 75 µM Gag-NtD (black) and from 75 µM equimolar mixtures of Gag-NtDs with Env(1–20) (red) are shown in each panel. (F) Histogram of equilibrium association constants derived from the sedimentation data as described in methods.

Mentions: In order to probe the importance of the interactions in the Gag-Env interface observed in the crystal structure a series of serine and asparagine substitutions were introduced at Val14, Leu17, Val18 and Leu21 to make the Env binding site progressively polar. In addition, in order to examine the contribution of the α1-β1 loop to the Env interaction a conservative Asn29 to Gln substitution was also introduced. The affinity of binding of these Gag-NtD mutants to Env-LP was examined using the sedimentation velocity assay, Figure 5A–E and Supplementary Figure S2. In all cases, the single polar substitutions introduced into the Env binding site reduced the affinity of the Gag-Env interaction. The decrease varied from 5 – 2 fold in the order Leu21>Val14>Leu17≈Val18 identifying these residues as being required for the Gag-Env interaction. Double substitutions decreased the affinity even further with the Val14/Leu21 to serine having the greatest effect, resulting in around a twenty-fold reduction in binding, Figure 5F. Moreover, the triple substitution where Val14, Val18 and Leu21 were all substituted by serine reduced binding to an undetectable level, Supplementary Figure S2. The conservative change Asn29 to glutamine has little effect on Env binding perhaps reflecting the importance of the backbone movement around Pro30 rather than sidechain interactions for Env-binding at this position.


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)

Sedimentation velocity analysis of Gag-Env interface mutants.C(S) functions that best fit sedimentation velocity profiles from (A) wt PFV-Gag-NtD, (B) V14S, (C) L17S, (D) V14S/L21S and (E) N29Q mutants. The C(S) function from 75 µM Gag-NtD (black) and from 75 µM equimolar mixtures of Gag-NtDs with Env(1–20) (red) are shown in each panel. (F) Histogram of equilibrium association constants derived from the sedimentation data as described in methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649970&req=5

ppat-1003376-g005: Sedimentation velocity analysis of Gag-Env interface mutants.C(S) functions that best fit sedimentation velocity profiles from (A) wt PFV-Gag-NtD, (B) V14S, (C) L17S, (D) V14S/L21S and (E) N29Q mutants. The C(S) function from 75 µM Gag-NtD (black) and from 75 µM equimolar mixtures of Gag-NtDs with Env(1–20) (red) are shown in each panel. (F) Histogram of equilibrium association constants derived from the sedimentation data as described in methods.
Mentions: In order to probe the importance of the interactions in the Gag-Env interface observed in the crystal structure a series of serine and asparagine substitutions were introduced at Val14, Leu17, Val18 and Leu21 to make the Env binding site progressively polar. In addition, in order to examine the contribution of the α1-β1 loop to the Env interaction a conservative Asn29 to Gln substitution was also introduced. The affinity of binding of these Gag-NtD mutants to Env-LP was examined using the sedimentation velocity assay, Figure 5A–E and Supplementary Figure S2. In all cases, the single polar substitutions introduced into the Env binding site reduced the affinity of the Gag-Env interaction. The decrease varied from 5 – 2 fold in the order Leu21>Val14>Leu17≈Val18 identifying these residues as being required for the Gag-Env interaction. Double substitutions decreased the affinity even further with the Val14/Leu21 to serine having the greatest effect, resulting in around a twenty-fold reduction in binding, Figure 5F. Moreover, the triple substitution where Val14, Val18 and Leu21 were all substituted by serine reduced binding to an undetectable level, Supplementary Figure S2. The conservative change Asn29 to glutamine has little effect on Env binding perhaps reflecting the importance of the backbone movement around Pro30 rather than sidechain interactions for Env-binding at this position.

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