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HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358.

Bichel K, Price AJ, Schaller T, Towers GJ, Freund SM, James LC - Retrovirology (2013)

Bottom Line: Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells.NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid.Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein and Nucleic Acid Chemistry Division, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

ABSTRACT

Background: Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown.

Results: Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection.

Conclusion: Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.

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NUP358Cyp catalyses cis-trans isomerization of HIV-1 CAN. (a-c) 2D 1H-15N ZZ-exchange spectra of HIV-1 CAN focused on Gly89, without enzyme (a), in the presence of NUP358Cyp (b) or CypA (c). Gly89 cis,trans1H,15N correlation or “auto” peaks are labeled and are the result of cis and trans forms of the proceeding Pro90 residue being populated at equilibrium. Addition of either NUP358Cyp or CypA yields exchange peaks that connect cis and trans auto peaks (broken lines). The cis exchange peak appears at the same 1H (15N) chemical shift position as the trans auto peak and vice versa. Note, that the trans exchange peak is largely obscured by additional signals in the spectra. (d-e) The intensities of both auto and exchange peaks vary as a result of the ZZ-mixing time (Tm). Fits of CypA (d) and NUP358Cyp (e) yield exchange constants of 4.3 and 12.1 s-1 respectively.
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Figure 3: NUP358Cyp catalyses cis-trans isomerization of HIV-1 CAN. (a-c) 2D 1H-15N ZZ-exchange spectra of HIV-1 CAN focused on Gly89, without enzyme (a), in the presence of NUP358Cyp (b) or CypA (c). Gly89 cis,trans1H,15N correlation or “auto” peaks are labeled and are the result of cis and trans forms of the proceeding Pro90 residue being populated at equilibrium. Addition of either NUP358Cyp or CypA yields exchange peaks that connect cis and trans auto peaks (broken lines). The cis exchange peak appears at the same 1H (15N) chemical shift position as the trans auto peak and vice versa. Note, that the trans exchange peak is largely obscured by additional signals in the spectra. (d-e) The intensities of both auto and exchange peaks vary as a result of the ZZ-mixing time (Tm). Fits of CypA (d) and NUP358Cyp (e) yield exchange constants of 4.3 and 12.1 s-1 respectively.

Mentions: The cyclophilin domain of NUP358 has been shown to be an active isomerase, although it possesses much weaker catalytic activity than CypA when tested using a synthetic proline-containing peptide substrate [21]. So far, the only biological role assigned for NUP358Cyp has been to facilitate the interconversion of thermodynamically or kinetically trapped isoforms of red/green opsin in cone cells via cis-trans prolyl isomerization of proline residues within opsin [28]. Given that CypA has been shown to isomerize HIV-1 capsid [9], we investigated whether CAN is also a substrate for NUP358Cyp using NMR ZZ-exchange spectroscopy. Previous work by Bosco et al. and Eisenmesser et al. has shown that ZZ-exchange is an effective way of measuring CypA isomerization under steady state conditions [9,29]. 2D 1H-15N ZZ-exchange data on uniformly 15N-labeled CAN were collected in the presence of NUP358Cyp and CypA and compared to intrinsic ZZ-exchange of 15N-labeled CAN alone. 1H-15N correlation spectra require amide protons and therefore proline residues (such as P90) are not detected. However, the adjacent residue, G89 is characterised by two 1H-15N correlation peaks indicating that the proceeding proline must exist in both cis and trans forms (Figure 3a-c). These “auto” peaks are detected in the absence or presence of NUP358Cyp or CypA, respectively. An estimation of the cis/trans distribution at equilibrium using 1H-15N correlation (HSQC) spectra revealed that ~14% of the capsid G89-P90 bond exists in cis and ~86% trans, as previously observed [9]. The introduction of a variable mixing period in ZZ-exchange experiments enables inter-conversion between the cis and trans isomers resulting in the mixing time dependent build up of “exchange” peaks which connect “auto” peaks in a distinct pattern. For CAN alone, there is no detectable magnetization transfer between the two species, as evidenced by the lack of “exchange” peaks at all time points. This suggests that uncatalyzed cis-trans isomerization is very slow, with an exchange rate < 0.1 s-1. However, addition of catalytic amounts (1:10 molar ratio) of either CypA or NUP358Cyp results in the rapid build-up of intense exchange peaks, indicating fast cis-trans isomerization is now taking place (Figure 3b,c). These experiments unambiguously confirm HIV-1 capsid as a substrate for NUP358Cyp isomerization.


HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358.

Bichel K, Price AJ, Schaller T, Towers GJ, Freund SM, James LC - Retrovirology (2013)

NUP358Cyp catalyses cis-trans isomerization of HIV-1 CAN. (a-c) 2D 1H-15N ZZ-exchange spectra of HIV-1 CAN focused on Gly89, without enzyme (a), in the presence of NUP358Cyp (b) or CypA (c). Gly89 cis,trans1H,15N correlation or “auto” peaks are labeled and are the result of cis and trans forms of the proceeding Pro90 residue being populated at equilibrium. Addition of either NUP358Cyp or CypA yields exchange peaks that connect cis and trans auto peaks (broken lines). The cis exchange peak appears at the same 1H (15N) chemical shift position as the trans auto peak and vice versa. Note, that the trans exchange peak is largely obscured by additional signals in the spectra. (d-e) The intensities of both auto and exchange peaks vary as a result of the ZZ-mixing time (Tm). Fits of CypA (d) and NUP358Cyp (e) yield exchange constants of 4.3 and 12.1 s-1 respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: NUP358Cyp catalyses cis-trans isomerization of HIV-1 CAN. (a-c) 2D 1H-15N ZZ-exchange spectra of HIV-1 CAN focused on Gly89, without enzyme (a), in the presence of NUP358Cyp (b) or CypA (c). Gly89 cis,trans1H,15N correlation or “auto” peaks are labeled and are the result of cis and trans forms of the proceeding Pro90 residue being populated at equilibrium. Addition of either NUP358Cyp or CypA yields exchange peaks that connect cis and trans auto peaks (broken lines). The cis exchange peak appears at the same 1H (15N) chemical shift position as the trans auto peak and vice versa. Note, that the trans exchange peak is largely obscured by additional signals in the spectra. (d-e) The intensities of both auto and exchange peaks vary as a result of the ZZ-mixing time (Tm). Fits of CypA (d) and NUP358Cyp (e) yield exchange constants of 4.3 and 12.1 s-1 respectively.
Mentions: The cyclophilin domain of NUP358 has been shown to be an active isomerase, although it possesses much weaker catalytic activity than CypA when tested using a synthetic proline-containing peptide substrate [21]. So far, the only biological role assigned for NUP358Cyp has been to facilitate the interconversion of thermodynamically or kinetically trapped isoforms of red/green opsin in cone cells via cis-trans prolyl isomerization of proline residues within opsin [28]. Given that CypA has been shown to isomerize HIV-1 capsid [9], we investigated whether CAN is also a substrate for NUP358Cyp using NMR ZZ-exchange spectroscopy. Previous work by Bosco et al. and Eisenmesser et al. has shown that ZZ-exchange is an effective way of measuring CypA isomerization under steady state conditions [9,29]. 2D 1H-15N ZZ-exchange data on uniformly 15N-labeled CAN were collected in the presence of NUP358Cyp and CypA and compared to intrinsic ZZ-exchange of 15N-labeled CAN alone. 1H-15N correlation spectra require amide protons and therefore proline residues (such as P90) are not detected. However, the adjacent residue, G89 is characterised by two 1H-15N correlation peaks indicating that the proceeding proline must exist in both cis and trans forms (Figure 3a-c). These “auto” peaks are detected in the absence or presence of NUP358Cyp or CypA, respectively. An estimation of the cis/trans distribution at equilibrium using 1H-15N correlation (HSQC) spectra revealed that ~14% of the capsid G89-P90 bond exists in cis and ~86% trans, as previously observed [9]. The introduction of a variable mixing period in ZZ-exchange experiments enables inter-conversion between the cis and trans isomers resulting in the mixing time dependent build up of “exchange” peaks which connect “auto” peaks in a distinct pattern. For CAN alone, there is no detectable magnetization transfer between the two species, as evidenced by the lack of “exchange” peaks at all time points. This suggests that uncatalyzed cis-trans isomerization is very slow, with an exchange rate < 0.1 s-1. However, addition of catalytic amounts (1:10 molar ratio) of either CypA or NUP358Cyp results in the rapid build-up of intense exchange peaks, indicating fast cis-trans isomerization is now taking place (Figure 3b,c). These experiments unambiguously confirm HIV-1 capsid as a substrate for NUP358Cyp isomerization.

Bottom Line: Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells.NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid.Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein and Nucleic Acid Chemistry Division, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

ABSTRACT

Background: Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown.

Results: Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection.

Conclusion: Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.

Show MeSH
Related in: MedlinePlus