Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

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bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

Section: Stability Of Aav Capsidssupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Rayaprolu

Kruse

Kant

et al. 2013

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108

“…The PLA2 domain is not exposed in assembled, full parvoviruses such as minute virus of mice (MVM) (13) and human parvovirus B19 (14), and it therefore has to be exposed during endocytosis (9,11,(13)(14)(15). However, the mechanism by which the VP1 amino-terminal PLA2 domain is exposed has not been elucidated in detail (16).…”

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confidence: 99%

The Structure and Host Entry of an Invertebrate Parvovirus

Meng

Zhang

Plevka

et al. 2013

bThe 3.5-Å resolution X-ray crystal structure of mature cricket parvovirus (Acheta domesticus densovirus [AdDNV]) has been determined. Structural comparisons show that vertebrate and invertebrate parvoviruses have evolved independently, although there are common structural features among all parvovirus capsid proteins. It was shown that raising the temperature of the AdDNV particles caused a loss of their genomes. The structure of these emptied particles was determined by cryo-electron microscopy to 5.5-Å resolution, and the capsid structure was found to be the same as that for the full, mature virus except for the absence of the three ordered nucleotides observed in the crystal structure. The viral protein 1 (VP1) amino termini could be externalized without significant damage to the capsid. In vitro, this externalization of the VP1 amino termini is accompanied by the release of the viral genome. Parvoviruses are small (ϳ250-to 300-Å-diameter), singlestranded DNA (ssDNA), icosahedral (Tϭ1), nonenveloped viruses whose genomes are approximately 5 kb long (1). The Parvoviridae family has been subdivided into viruses that infect vertebrates (Parvovirinae) and those that infect invertebrates (Densovirinae) (2). Parvoviruses replicate in dividing cells such as in tissues from insect larvae and fetuses. Densoviruses are highly pathogenic, and those that use insect hosts usually kill 90% of the larvae within a few days (2). Densoviruses pose a threat to commercial invertebrates such as shrimp (3), silkworms (4), and crickets (5, 6). Some highly pathogenic densoviruses are potential selective pesticides for vectors that transmit mosquito-borne diseases (7). Parvovirinae generally have three types of proteins (VP1, VP2, and VP3) in their capsids (8), whereas Densovirinae generally have four types of proteins (VP1 to VP4) in their capsids (2). In densoviruses there are 200 additional amino acids in VP1 at the N terminus. These different proteins result from different initiation sites for translation of the capsid gene and from posttranslational modification of their N termini (8). Generally, each of the 60 subunits within a capsid has the same amino acid sequence and is structurally the same, except that the different proteins start at different amino acids. The VP2s of some densoviruses are unique among VP2s of parvoviruses since they are not completely contained within corresponding VP1s (Fig. 1A).Parvoviruses enter cells by dynamin-dependent receptor-mediated endocytosis and escape the endosome by the phospholipase (PLA2) activity within the amino-terminal domain of VP1 (9-13). Although there is often less than 5% amino acid identity among the structural proteins of parvoviruses, the sequence of the PLA2 N-terminal domain of VP1 has more than 30% amino acid identity ( Fig. 1A and B). The PLA2 domain is not exposed in assembled, full parvoviruses such as minute virus of mice (MVM) (13) and human parvovirus B19 (14), and it therefore has to be exposed during endocytosis (9,11,(13)(14)(15). However, the mechanism by which ...

“…While virions are in the endosomal pathway, acidification and translocation to late endosomes are required (9). This environment triggers conformational changes in the virion, leading to externalization of the N terminus of capsid proteins (10). PPV capsids consist of two proteins.…”

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confidence: 99%

“…The VP1 sequence comprises the VP2 sequence with a 150-amino-acid N-terminal extension, called VP1up, for VP1 unique part (15). VP1up externalization in the endosomes is absolutely required for PPV infection since it contains the phospholipase A 2 (PLA 2 ) activity, essential to breach the endosomal membrane barrier (10,16,17).…”

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confidence: 99%

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

Boisvert

Bouchard-Lévesque

Fernandes

et al. 2014

Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCEMost DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.