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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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Related in: MedlinePlus

Conformation and solution oligomeric state of FV-Gag-NtDs.(A) SEC-MALLS analysis of PFV-Gag-NtD recorded at 2.5 mgml−1 (red), 5.0 mgml−1 (blue), 7.5 mgml−1 (green) and 10.0 mgml−1 (pink) (left panel) and FFV Gag NtD at 1.5 mgml−1 (red) 3.0 mgml−1 (blue), 6.0 mgml−1 (green) and 12.5 mgml−1 (pink) (Right panel). Differential refractive index (dRI) is plotted against retention time and the molar mass distributions, determined throughout the elution of each peak, plotted as points. (B) C(S) distributions derived from sedimentation velocity data recorded from PFV-Gag-NtD at 0.5 mgml−1 (red), 1.0 mgml−1 (blue) and 2.0 mgml−1 (green) (left panel) and FFV-Gag-NtD at 0.6 mgml−1 (red), 2.0 mgml−1 (blue) and 2.5 mgml−1 (green) (right panel). (C) Multi-speed sedimentation equilibrium profiles determined from interference data collected on PFV-Gag-NtD at 67 µM (left panel) and FFV-Gag-NtD at 50 µM (right panel). Data was recorded at the three speeds indicated. The solid lines represent the best fit to the data using a single species model.
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ppat-1003376-g002: Conformation and solution oligomeric state of FV-Gag-NtDs.(A) SEC-MALLS analysis of PFV-Gag-NtD recorded at 2.5 mgml−1 (red), 5.0 mgml−1 (blue), 7.5 mgml−1 (green) and 10.0 mgml−1 (pink) (left panel) and FFV Gag NtD at 1.5 mgml−1 (red) 3.0 mgml−1 (blue), 6.0 mgml−1 (green) and 12.5 mgml−1 (pink) (Right panel). Differential refractive index (dRI) is plotted against retention time and the molar mass distributions, determined throughout the elution of each peak, plotted as points. (B) C(S) distributions derived from sedimentation velocity data recorded from PFV-Gag-NtD at 0.5 mgml−1 (red), 1.0 mgml−1 (blue) and 2.0 mgml−1 (green) (left panel) and FFV-Gag-NtD at 0.6 mgml−1 (red), 2.0 mgml−1 (blue) and 2.5 mgml−1 (green) (right panel). (C) Multi-speed sedimentation equilibrium profiles determined from interference data collected on PFV-Gag-NtD at 67 µM (left panel) and FFV-Gag-NtD at 50 µM (right panel). Data was recorded at the three speeds indicated. The solid lines represent the best fit to the data using a single species model.

Mentions: Given the unexpected nature of the dimer observed in the crystal structure, the conformation and self-association properties of the Gag-NtD from PFV and from the non-primate feline foamy virus (FFV) were examined using a variety of solution hydrodynamic methods. Initial assessment by Size Exclusion Chromatography coupled Multi-Angle Laser Light Scattering (SEC-MALLS), over a range of protein concentration (12-1.5 mg/ml), yielded invariant solution molecular weights of 40.0 kDa and 34.0 kDa for PFV- and FFV-Gag-NtD respectively, Figure 2A. By comparison, the sequence-derived molecular weights are 22.8 kDa and 19.0 kDa. Given these values together with the lack of a concentration dependency of the molecular weight it is apparent that along with PFV, the Gag-NtD from FFV also forms strong dimers in solution. To confirm the oligomeric state, velocity (SV-AUC) and equilibrium (SE-AUC) analytical ultracentrifugation of PFV- and FFV-Gag-NtD was undertaken. A summary of the experimental parameters, molecular weights derived from the data and statistics relating to the quality of fits are shown in Table 2. Analysis of the sedimentation velocity data for PFV-Gag-NtD revealed no concentration dependency of the sedimentation coefficient (S20,w = 3.08) over the range measured, Figure 2B. Similar data were obtained for FFV-Gag-NtD (S20,w = 2.72) indicating both proteins are single stable species. The molecular weights derived from either C(S) or discrete component analysis were 47 kD and 36 kD respectively, Table 2, consistent with a PFV- and FFV-Gag-NtD dimer. The frictional ratios (f/fo) obtained from the analysis, 1.4–1.5, also suggest both dimers have a similar elongated conformation. Multispeed sedimentation equilibrium studies at varying initial protein concentration were also carried out and typical equilibrium distributions for PFV- and FFV-Gag-NtD from individual multispeed experiments are presented in Figure 2C. Analysis of individual gradient profiles showed no concentration dependency of the molecular weight and so data were fit globally with a single ideal molecular species model, producing weight averaged molecular weights of 44 kDa and 33.7 kDa for PFV- and FFV-Gag-NtD respectively. These data confirm that formation of stable dimeric structures is a common property shared among the Gag proteins of divergent FVs and N-terminal domain mediated dimerisation is likely an important component of FV assembly.


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)

Conformation and solution oligomeric state of FV-Gag-NtDs.(A) SEC-MALLS analysis of PFV-Gag-NtD recorded at 2.5 mgml−1 (red), 5.0 mgml−1 (blue), 7.5 mgml−1 (green) and 10.0 mgml−1 (pink) (left panel) and FFV Gag NtD at 1.5 mgml−1 (red) 3.0 mgml−1 (blue), 6.0 mgml−1 (green) and 12.5 mgml−1 (pink) (Right panel). Differential refractive index (dRI) is plotted against retention time and the molar mass distributions, determined throughout the elution of each peak, plotted as points. (B) C(S) distributions derived from sedimentation velocity data recorded from PFV-Gag-NtD at 0.5 mgml−1 (red), 1.0 mgml−1 (blue) and 2.0 mgml−1 (green) (left panel) and FFV-Gag-NtD at 0.6 mgml−1 (red), 2.0 mgml−1 (blue) and 2.5 mgml−1 (green) (right panel). (C) Multi-speed sedimentation equilibrium profiles determined from interference data collected on PFV-Gag-NtD at 67 µM (left panel) and FFV-Gag-NtD at 50 µM (right panel). Data was recorded at the three speeds indicated. The solid lines represent the best fit to the data using a single species model.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003376-g002: Conformation and solution oligomeric state of FV-Gag-NtDs.(A) SEC-MALLS analysis of PFV-Gag-NtD recorded at 2.5 mgml−1 (red), 5.0 mgml−1 (blue), 7.5 mgml−1 (green) and 10.0 mgml−1 (pink) (left panel) and FFV Gag NtD at 1.5 mgml−1 (red) 3.0 mgml−1 (blue), 6.0 mgml−1 (green) and 12.5 mgml−1 (pink) (Right panel). Differential refractive index (dRI) is plotted against retention time and the molar mass distributions, determined throughout the elution of each peak, plotted as points. (B) C(S) distributions derived from sedimentation velocity data recorded from PFV-Gag-NtD at 0.5 mgml−1 (red), 1.0 mgml−1 (blue) and 2.0 mgml−1 (green) (left panel) and FFV-Gag-NtD at 0.6 mgml−1 (red), 2.0 mgml−1 (blue) and 2.5 mgml−1 (green) (right panel). (C) Multi-speed sedimentation equilibrium profiles determined from interference data collected on PFV-Gag-NtD at 67 µM (left panel) and FFV-Gag-NtD at 50 µM (right panel). Data was recorded at the three speeds indicated. The solid lines represent the best fit to the data using a single species model.
Mentions: Given the unexpected nature of the dimer observed in the crystal structure, the conformation and self-association properties of the Gag-NtD from PFV and from the non-primate feline foamy virus (FFV) were examined using a variety of solution hydrodynamic methods. Initial assessment by Size Exclusion Chromatography coupled Multi-Angle Laser Light Scattering (SEC-MALLS), over a range of protein concentration (12-1.5 mg/ml), yielded invariant solution molecular weights of 40.0 kDa and 34.0 kDa for PFV- and FFV-Gag-NtD respectively, Figure 2A. By comparison, the sequence-derived molecular weights are 22.8 kDa and 19.0 kDa. Given these values together with the lack of a concentration dependency of the molecular weight it is apparent that along with PFV, the Gag-NtD from FFV also forms strong dimers in solution. To confirm the oligomeric state, velocity (SV-AUC) and equilibrium (SE-AUC) analytical ultracentrifugation of PFV- and FFV-Gag-NtD was undertaken. A summary of the experimental parameters, molecular weights derived from the data and statistics relating to the quality of fits are shown in Table 2. Analysis of the sedimentation velocity data for PFV-Gag-NtD revealed no concentration dependency of the sedimentation coefficient (S20,w = 3.08) over the range measured, Figure 2B. Similar data were obtained for FFV-Gag-NtD (S20,w = 2.72) indicating both proteins are single stable species. The molecular weights derived from either C(S) or discrete component analysis were 47 kD and 36 kD respectively, Table 2, consistent with a PFV- and FFV-Gag-NtD dimer. The frictional ratios (f/fo) obtained from the analysis, 1.4–1.5, also suggest both dimers have a similar elongated conformation. Multispeed sedimentation equilibrium studies at varying initial protein concentration were also carried out and typical equilibrium distributions for PFV- and FFV-Gag-NtD from individual multispeed experiments are presented in Figure 2C. Analysis of individual gradient profiles showed no concentration dependency of the molecular weight and so data were fit globally with a single ideal molecular species model, producing weight averaged molecular weights of 44 kDa and 33.7 kDa for PFV- and FFV-Gag-NtD respectively. These data confirm that formation of stable dimeric structures is a common property shared among the Gag proteins of divergent FVs and N-terminal domain mediated dimerisation is likely an important component of FV assembly.

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