The Expression and Function of the Small Nonstructural Proteins of Adeno-Associated Viruses (AAVs)

Figure 3. The genetic maps of AAV2 and AAV5. Adapted from [58,65,70]. The ~4.7 kb ssDNA genomes of AAV2 (GenBank accession no. AF043303) (A) and AAV5 (AF085716) (B) are depicted with two ORFs (Rep and Cap); one intron that is spliced at one donor (D); two alternative acceptors, A1 as the minor and A2 the major sites, one (AAV2) or two (AAV5) polyadenylation sites (pA); and two ITRs. Three promoters, P5 (AAV2)/P7 (AAV5), P19, and P40, transcribe three sets of AAV mRNAs, P5/P7 mRNAs, P19 mRNAs, and P40/P41 mRNAs, respectively. For AAV2 (A), P5 mRNAs encode Rep78, Rep68m, and Rep68M; and P19 mRNAs encode Rep52, Rep40m, and Rep40M. For AAV5 (B), P7 mRNA encodes only Rep78, and P19 mRNA encodes Rep40. P40 (AAV2)/P41 (AAV5) mRNAs include P40 unspliced mRNA and alternatively spliced mRNAs at D and A1 or A2, designated as P40/P41-SplA1 and P40/P41-SplA2 mRNA, respectively. SplA1 mRNA encodes VP1, and SplA2 mRNA encodes MAAP, VP2, AAP, and VP3 at the initiation codons as indicated. MAAP and AAP are translated from the +1 frame-shifted ORF. Created in Biorender.com (accessed on 25 July 2024).

Figure 1. AAV life cycle. AAV is attached to the host cell via glycans. Internalization occurs by various endocytic pathways (clathrin-mediated endocytosis, CLIC/GEEC, or micropinocytosis). Internalized AAV is endocytosed in the early endosome, where the externalization of VP1u occurs. VP1u-externalized AAV then traffics towards the nucleus by following a path through the TGN, Golgi, and ER, or after cytoplasmic escape. Escaped AAV can be degraded after ubiquitination in the proteasome. Entry into the nucleus occurs via nuclear pore complexes (NPC). Once AAV is localized into the nucleus, the capsid disassembles, and viral proteins are expressed following transcription. Viral genome replication follows a rolling-hairpin DNA replication (RHR) model [47,48]. The ssDNA genome is extended from the 3′-OH of the ITR to form a monomer turnround replicative form (mtRF). The hairpin is nicked at a terminal resolution site (TRS) by Rep78/68. The hairpinned end is unwound, and the 3′-end formed by Rep cleavage is extended to the end of the template strand. The ends of each strand refold into their alternative self-base pairing hairpin structures, and the full-length DNA synthesis from the 3′-primer…

Figure 2. The ITR sequences and structures of AAV2 (A) and AAV5 (B). Both AAV2 and AAV5 ITRs are diagrammed in a “flip” orientation. The complementary A/A’, B/B’, and C/C’ sequences are labeled. The D-sequence is contiguous with the ssDNA genome body. The terminal resolution site (TRS) consists of the cleavage site (purple arrow) and the flanking nucleotides in the purple line box. Both the AAV2 and AAV5 Rep-binding elements (RBEs) consist of >(GAGC)3, as shown in the purple-lined box. In the ITRs of AAV1–4, 6, and 7 ITRs, multiple transcription start sites (TSSs), indicated by arrows, were found to cluster at the RBE [57]. The Inr of the AAV5 ITR has one TSS at nt 142 [58]. The AAV5 ITR is only 58% homologous with the AAV2 ITR.

Figure 4. Alignment of AAP sequences of AAV1–13. Predicted AAP protein sequences derived from +1 frame-shifted ORF in the Cap genes of AAV1–13 are aligned using MUSCLE and colored with the Clustal X scheme. Consensus amino acids are shown with a threshold of 50%. The CUG codon is conserved as the initiator of translation for all AAPs. Predicted AAP protein sequences are obtained from corresponding cap gene sequences as follows: AAV1-AF063497, AAV2-AF043303, AAV3B-AF028705, AAV4-NC_001829, AAV5-AF085716, AAV6-AF028704, AAV7-NC_006260, AAV8-AF513852, AAV9-AY530579, AAV10-AY631965, AAV11-AY631966, AAV12-DQ813647, and AAV13-EU285562. Domains or motifs of predicted amino acids are underlined. Threonine/serine, T/S, rich. Various motifs are predicted and indicated. Amino acids identical in all 13 proteins are indicated by (*), strongly similar amino acids by (:), and weakly similar by “(.)”.

Figure 5. Alignment of MAAP sequences of AAV1–13. Predicted MAAP protein sequences derived from +1 frame-shifted ORF in the Cap gene are aligned using MUSCLE and colored with the Clustal X scheme. Consensus amino acids are shown with a threshold of 50%. Predicted MAAP protein sequences are obtained from corresponding Cap gene sequences as follows: AAV1-AF063497, AAV2-AF043303, AAV3B-AF028705, AAV4-NC_001829, AAV5-AF085716, AAV6-AF028704, AAV7-NC_006260, AAV8-AF513852, AAV9-AY530579, AAV10-AY631965, AAV11-AY631966, AAV12-DQ813647, and AAV13-EU285562. Domains or motifs of predicted amino acids are underlined. Amino acids identical in all 13 proteins are indicated by (*), strongly similar amino acids by (:), and weakly similar by “(.)”.

Figure 6. Putative egress routes for AAV2 in nonlytic infection. Assembled AAV2 virions are exported from the nucleus to the endoplasmic reticulum (ER), and then reach the cis-Golgi Network (CGN) to the trans-Golgi Network (TGN), which may be mediated by COPII-coated vesicles, like during MVM egress [107]. In one way, virions are packaged in Rab8-positive cargos, which are directed to the plasma membrane for release as microvesicles. On the other hand, virions-containing vesicles can reach early endosomes marked with Rab11 and then traffic to recycling endosomes or late endosomes. The virions in the recycling endosomes can be released as microvesicles. The virions in late endosomes marked with Rab11 and CD63 can be released as exosomes. Rab7-marked late endosomes are led to lysosomes, which will later be degraded in acidified lysosomes or potentially released in deacidified lysosomes by lysosomal exocytosis. MAAP, as marked with stars, could be involved in each trafficking pathway from the nucleus to the plasma membrane as marked. MVB, multivesicular body. Created with BioRender.com (accessed on 25 July 2024).