LETTER
New structural model of adenoviral cement proteins is not yet concrete A recent reinterpretation of the refined crystal structure of human adenovirus type 5 (HAdV5) (1) has resulted in the reassignment of minor capsid/cement proteins IX and IIIa. The revised model differs substantially from the prior one, based on recent cryo-EM studies (2). Which model is correct? The answer may affect the assignment of proteins V, VI, and VIII and will affect our understanding of Ad structure and function. Although dispensable for assembly and infectivity, IX is a 14-kDa protein that stabilizes the Ad virion by cementing the “Group of Nine” (GON) hexons together on each facet. In the old model, the N terminus of IX folds into an extended “triskelion” structure with four triskelions in each of the 20 facets (240 molecules per virion). IX has a conserved C-terminal coiled coil (CC), separated from the triskelion by a linker (Fig. 1 A and B). In HAdV5, four CC domains from different triskelions associate into a unique four-helix bundle (4-HLXB), seen as elongated density bridging the interfacet hexons. In this peculiar arrangement, one helix originates from a triskelion of the neighboring facet, running antiparallel to the other three (Fig. 1C). IIIa is a 65-kDa protein present at 60 copies per virion that is essential for genome packaging and maturation. Virus lacking IIIa only assembles empty immature particles. Evidence suggests IIIa spans the capsid with
regions exposed on the surface, yet IIIa interacts with core protein VII (3). The previous model had the bulk of IIIa at the vertices, underneath the penton base. The new model has the direction of the IX polypeptide chain reversed and has reassigned the 4-HLXB density as protein IIIa residues 103–342 (with an unresolved loop from 201 to 261). Two major discrepancies should be addressed before this new model can be accepted. The first problem arises from cryo-EM studies of bovine and canine types (BAdV3 and CAdV2) (4), revealing that rather than looping around neighboring hexons to form the 4-HLXB density, the IX CC domain forms a trimeric bundle immediately above the triskelions (Fig. 1D). This result is attributed to the shorter linkers of these types, precluding the CC domains from achieving the conformation seen with HAdV5 (Fig. 1A). The 4-HLXB density is notably absent in these animal types (Fig. 1D). Given that IIIa is highly conserved, particularly in the residues assigned to the 4-HLXB (Fig. 1E), then where is the 4-HLXB density? Why would IIIa occupy a different locale in animal types? Second, chain reversal of IX has the triskelion core being formed by an Ala/Ser-rich region of the linker (residues 60–70), which is also reported to mediate interactions between the hexon β-barrels (1). This result is inconsistent with work showing that mutant
E4542–E4543 | PNAS | October 28, 2014 | vol. 111 | no. 43
HAdV5 with IX deleted for residues 60–72 behaves like wild-type virus in terms of encapsidation of IX and the associated thermostability phenotype (5). These discrepancies need to be resolved before the new model can be accepted. Perhaps structural studies of ΔIIIa particles will clarify the confusion over IX and IIIa. Samuel K. Campos1 Department of Immunobiology, Department of Molecular & Cellular Biology, and BIO5 Institute, University of Arizona, Tucson, AZ 85721 1 Reddy VS, Nemerow GR (2014) Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection. Proc Natl Acad Sci USA 111(32):11715–11720. 2 Liu H, et al. (2010) Atomic structure of human adenovirus by cryoEM reveals interactions among protein networks. Science 329(5995): 1038–1043. 3 Boudin ML, D’Halluin JC, Cousin C, Boulanger P (1980) Human adenovirus type 2 protein IIIa. II. Maturation and encapsidation. Virology 101(1):144–156. 4 Cheng L, et al. (2014) Cryo-EM structures of two bovine adenovirus type 3 intermediates. Virology 450-451:174–181. 5 Vellinga J, van den Wollenberg DJ, van der Heijdt S, Rabelink MJ, Hoeben RC (2005) The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability. J Virol 79(5):3206–3210.
Author contributions: S.K.C. analyzed data and wrote the paper. The author declares no conflict of interest. 1
Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1415364111
LETTER
A
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C P H1 H4
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IIIa HAdV5 103-342 A R Y N S G N V Q T N L D R L V G D V R E A V A Q R E R A Q Q Q G N L G S MV A L N A F L S T Q P A N V P R G Q E D Y T N F V S A L R L MV T E T P Q S E V Y Q IIIa BAdV3 124-363 A R Y N S V N V Q G N L D R L I Q D V K E A L A Q R E R T G P GA G L G S V V A L N A F L S T Q P A V V E R G Q E N Y V A F V S A L K L MV T E A P Q S E V Y Q IIIa CAdV2 114-352 A K Y N S L N A Q S N L E R L A G D V R E A V A Q QV R I A T G - N L G S L T A L N G F L A R L P A N V E R G Q E N Y T G F V S A L K L L V S E V P S T E V Y Q
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IIIa HAdV5 103-342 S G P D Y F F Q T S R Q G L Q T V N L S QA F K N L Q G LWGV R A P T G D R A T V S S L L T P N S R L L L L L I A P F T D S G S V S R D T Y L G H L L T L Y R IIIa BAdV3 124-363 A G P S F F F Q T S R H G S Q T V N L S QA F D N L R P LWGV R A P V H E R T T I S S L L T P N T R L L L L L I A P F T D S V G I S R D S Y L G H L L T L Y R IIIa CAdV2 114-352 S G P H Y F L Q S S R N G T Q T V N L T N A F E N L K P LWGV K A P T M E R L S I S A L L T P N T R L L L L L V S P F T D S V S I S R D S Y L G Y L L T L Y R
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IIIa HAdV5 103-342 E A I G QA H V D E H T F Q E I T S V S R A L G Q E D T G S L E A T L N Y L L T N R R Q K I P S L H S L N S E E E R I L R Y V Q Q S V S L N L MR D GV T P S V IIIa BAdV3 124-363 E T I G N T R V D E T T Y N E I T E V S R A L GA E D A S N L QA T L N Y L L T N K Q S K L P Q E F S L S P E E E R V L R Y V Q Q S V S L F L MQ D G H T A T T IIIa CAdV2 114-352 E A L G R N H L D E R T L E E V T E V S R A MG S E N I N N L QA T L N F L L T N R Q K R I P K D Y S L T P E E E R I V R F V Q QA V S L R MMQ E N L S P T E
Fig. 1. (A) Schematic diagram of IX from human (HAdV5) and animal (BAdV3, CAdV2) types. Note the difference in linker length between HAdV5 and animal types. (B) The CC domain is well conserved across types, with a predominance of hydrophobic residues at the a and d positions of the heptad repeat. (C) Schematic of the position of IX within a facet of the HAdV5 capsid. The red oval highlights the position of the 4-HLXB density in the center of an asymmetric unit composed of hexons H3/H4 and H1′/H2′ of the adjacent facet. This density is assigned as the four IX CCs in ref. 2 and as IIIa in ref. 1. The fourth CC domain from the neighboring facet triskelion is shown for clarity. (Inset) The density map (PDB ID code 3IYN) from ref. 2, highlighting the 4-HLXB density in magenta. (D) Schematic of a facet from BAdV3 highlighting the lack of 4-HLXB density in the red oval. Instead, the IX CCs are positioned above the triskelions, because of the shorter linkers. (Inset) The density map (PDB ID code 3ZIF) from ref. 4, again lacking 4-HLXB density but showing the IX CC domains in magenta. (E) Sequence alignment of the portion of IIIa assigned as the 4-HLXB density. Note the very high conservation across human, bovine, and canine types.
Campos
PNAS | October 28, 2014 | vol. 111 | no. 43 | E4543
New structural model of adenoviral cement proteins is not yet concrete A recent reinterpretation of the refined crystal structure of human adenovirus type 5 (HAdV5) (1) has resulted in the reassignment of minor capsid/cement proteins IX and IIIa. The revised model differs substantially from the prior one, based on recent cryo-EM studies (2). Which model is correct? The answer may affect the assignment of proteins V, VI, and VIII and will affect our understanding of Ad structure and function. Although dispensable for assembly and infectivity, IX is a 14-kDa protein that stabilizes the Ad virion by cementing the “Group of Nine” (GON) hexons together on each facet. In the old model, the N terminus of IX folds into an extended “triskelion” structure with four triskelions in each of the 20 facets (240 molecules per virion). IX has a conserved C-terminal coiled coil (CC), separated from the triskelion by a linker (Fig. 1 A and B). In HAdV5, four CC domains from different triskelions associate into a unique four-helix bundle (4-HLXB), seen as elongated density bridging the interfacet hexons. In this peculiar arrangement, one helix originates from a triskelion of the neighboring facet, running antiparallel to the other three (Fig. 1C). IIIa is a 65-kDa protein present at 60 copies per virion that is essential for genome packaging and maturation. Virus lacking IIIa only assembles empty immature particles. Evidence suggests IIIa spans the capsid with
regions exposed on the surface, yet IIIa interacts with core protein VII (3). The previous model had the bulk of IIIa at the vertices, underneath the penton base. The new model has the direction of the IX polypeptide chain reversed and has reassigned the 4-HLXB density as protein IIIa residues 103–342 (with an unresolved loop from 201 to 261). Two major discrepancies should be addressed before this new model can be accepted. The first problem arises from cryo-EM studies of bovine and canine types (BAdV3 and CAdV2) (4), revealing that rather than looping around neighboring hexons to form the 4-HLXB density, the IX CC domain forms a trimeric bundle immediately above the triskelions (Fig. 1D). This result is attributed to the shorter linkers of these types, precluding the CC domains from achieving the conformation seen with HAdV5 (Fig. 1A). The 4-HLXB density is notably absent in these animal types (Fig. 1D). Given that IIIa is highly conserved, particularly in the residues assigned to the 4-HLXB (Fig. 1E), then where is the 4-HLXB density? Why would IIIa occupy a different locale in animal types? Second, chain reversal of IX has the triskelion core being formed by an Ala/Ser-rich region of the linker (residues 60–70), which is also reported to mediate interactions between the hexon β-barrels (1). This result is inconsistent with work showing that mutant
E4542–E4543 | PNAS | October 28, 2014 | vol. 111 | no. 43
HAdV5 with IX deleted for residues 60–72 behaves like wild-type virus in terms of encapsidation of IX and the associated thermostability phenotype (5). These discrepancies need to be resolved before the new model can be accepted. Perhaps structural studies of ΔIIIa particles will clarify the confusion over IX and IIIa. Samuel K. Campos1 Department of Immunobiology, Department of Molecular & Cellular Biology, and BIO5 Institute, University of Arizona, Tucson, AZ 85721 1 Reddy VS, Nemerow GR (2014) Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection. Proc Natl Acad Sci USA 111(32):11715–11720. 2 Liu H, et al. (2010) Atomic structure of human adenovirus by cryoEM reveals interactions among protein networks. Science 329(5995): 1038–1043. 3 Boudin ML, D’Halluin JC, Cousin C, Boulanger P (1980) Human adenovirus type 2 protein IIIa. II. Maturation and encapsidation. Virology 101(1):144–156. 4 Cheng L, et al. (2014) Cryo-EM structures of two bovine adenovirus type 3 intermediates. Virology 450-451:174–181. 5 Vellinga J, van den Wollenberg DJ, van der Heijdt S, Rabelink MJ, Hoeben RC (2005) The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability. J Virol 79(5):3206–3210.
Author contributions: S.K.C. analyzed data and wrote the paper. The author declares no conflict of interest. 1
Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1415364111
LETTER
A
B
C P H1 H4
P
H2
H4
H3
H3
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P
D P H1 H4
H2
H4
H3
H3
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IIIa HAdV5 103-342 A R Y N S G N V Q T N L D R L V G D V R E A V A Q R E R A Q Q Q G N L G S MV A L N A F L S T Q P A N V P R G Q E D Y T N F V S A L R L MV T E T P Q S E V Y Q IIIa BAdV3 124-363 A R Y N S V N V Q G N L D R L I Q D V K E A L A Q R E R T G P GA G L G S V V A L N A F L S T Q P A V V E R G Q E N Y V A F V S A L K L MV T E A P Q S E V Y Q IIIa CAdV2 114-352 A K Y N S L N A Q S N L E R L A G D V R E A V A Q QV R I A T G - N L G S L T A L N G F L A R L P A N V E R G Q E N Y T G F V S A L K L L V S E V P S T E V Y Q
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IIIa HAdV5 103-342 S G P D Y F F Q T S R Q G L Q T V N L S QA F K N L Q G LWGV R A P T G D R A T V S S L L T P N S R L L L L L I A P F T D S G S V S R D T Y L G H L L T L Y R IIIa BAdV3 124-363 A G P S F F F Q T S R H G S Q T V N L S QA F D N L R P LWGV R A P V H E R T T I S S L L T P N T R L L L L L I A P F T D S V G I S R D S Y L G H L L T L Y R IIIa CAdV2 114-352 S G P H Y F L Q S S R N G T Q T V N L T N A F E N L K P LWGV K A P T M E R L S I S A L L T P N T R L L L L L V S P F T D S V S I S R D S Y L G Y L L T L Y R
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IIIa HAdV5 103-342 E A I G QA H V D E H T F Q E I T S V S R A L G Q E D T G S L E A T L N Y L L T N R R Q K I P S L H S L N S E E E R I L R Y V Q Q S V S L N L MR D GV T P S V IIIa BAdV3 124-363 E T I G N T R V D E T T Y N E I T E V S R A L GA E D A S N L QA T L N Y L L T N K Q S K L P Q E F S L S P E E E R V L R Y V Q Q S V S L F L MQ D G H T A T T IIIa CAdV2 114-352 E A L G R N H L D E R T L E E V T E V S R A MG S E N I N N L QA T L N F L L T N R Q K R I P K D Y S L T P E E E R I V R F V Q QA V S L R MMQ E N L S P T E
Fig. 1. (A) Schematic diagram of IX from human (HAdV5) and animal (BAdV3, CAdV2) types. Note the difference in linker length between HAdV5 and animal types. (B) The CC domain is well conserved across types, with a predominance of hydrophobic residues at the a and d positions of the heptad repeat. (C) Schematic of the position of IX within a facet of the HAdV5 capsid. The red oval highlights the position of the 4-HLXB density in the center of an asymmetric unit composed of hexons H3/H4 and H1′/H2′ of the adjacent facet. This density is assigned as the four IX CCs in ref. 2 and as IIIa in ref. 1. The fourth CC domain from the neighboring facet triskelion is shown for clarity. (Inset) The density map (PDB ID code 3IYN) from ref. 2, highlighting the 4-HLXB density in magenta. (D) Schematic of a facet from BAdV3 highlighting the lack of 4-HLXB density in the red oval. Instead, the IX CCs are positioned above the triskelions, because of the shorter linkers. (Inset) The density map (PDB ID code 3ZIF) from ref. 4, again lacking 4-HLXB density but showing the IX CC domains in magenta. (E) Sequence alignment of the portion of IIIa assigned as the 4-HLXB density. Note the very high conservation across human, bovine, and canine types.
Campos
PNAS | October 28, 2014 | vol. 111 | no. 43 | E4543
