Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene ( cap )-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans . In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Adeno-associated viruses (AAVs) are small, non-enveloped viruses that package a single-stranded (ss)DNA genome of 4.7 kilobases (kb) within their T = 1 icosahedral capsid. AAVs are replication-deficient viruses that require a helper virus to complete their life cycle. Recombinant (r)AAVs have been utilized as gene delivery vectors for decades in gene therapy applications. So far, six rAAV-based gene medicines have been approved by the US FDA. The 4.7 kb ssDNA genome of AAV encodes nine proteins, including three viral structural/capsid proteins, VP1, VP2, and VP3; four large nonstructural proteins (replication-related proteins), Rep78/68 and Rep52/40; and two small nonstructural proteins. The two nonstructured proteins are viral accessory proteins, namely the assembly associated protein (AAP) and membrane-associated accessory protein (MAAP). Although the accessory proteins are conserved within AAV serotypes, their functions are largely obscure. In this review, we focus on the expression strategy and functional properties of the small nonstructural proteins of AAVs.

We conducted a genome-wide CRISPR/Cas9 screen in suspension 293 F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screening included the previously reported KIAA0319L , TM9SF2 , and RNF121 , along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization, and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 ( SLC35A1 ), a Golgi apparatus-localized cytidine 5’-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, was only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3, which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (∆C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting that the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization. IMPORTANCE: rAAV is an essential tool for gene delivery in the treatment of genetic disorders; however, the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293 F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 10 ⁵ virions per cm ² of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.

Parvovirus B19 (B19V), a member of the genus Erythroparvovirus within the Parvoviridae family, infects human erythroid progenitor cells (EPCs) of bone marrow and fetal liver and causes various hematological disorders. The minor capsid protein VP1 of B19V contains a unique N-terminal region (VP1u) that facilitates virus binding and internalization into EPCs via its receptor-binding domain (RBD). We previously identified tyrosine-protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. In this study, we employed an ascorbate peroxidase 2 (APEX2)-based proximity labeling method to identify host proteins that are associated with B19V VP1u during entry. This analysis revealed human transferrin receptor 1 (hTfR) as a key host protein associated with VP1u. hTfR knockdown in UT7/Epo-S1 cells, a B19V-permissive human megakaryoblastoid leukemia cell line, showed significantly reduced B19V internalization and replication. Biolayer interferometry (BLI) assays confirmed a direct interaction between B19V VP1u and hTfR extracellular domain (ECD). Inhibition of VP1u interaction with hTfR ECD, either by a monoclonal antibody targeting the apical domain of the ECD or human ferritin, a natural ligand of hTfR that binds the apical domain, significantly reduced VP1u binding to hTfR, as well as B19V internalization and B19V replication in ex vivo -expanded EPCs. Furthermore, mutant RBD proteins that bear amino acid substitutions in the three helical domains nearly abolished RBD binding to hTfR and significantly reduced the ability to inhibit B19V infection of EPCs. Collectively, our findings establish hTfR as a B19V entry co-receptor that mediates B19V internalization into its natural host EPCs. IMPORTANCE: B19V causes severe hematological disorders, including transient aplastic crisis, chronic pure red cell aplasia, and hydrops fetalis, by selectively infecting erythroid progenitor cells (EPCs). Despite its clinical impact, no approved antivirals or vaccines exist, largely due to limited understanding of viral entry mechanisms. A unique feature of B19V is the externalization of the VP1 unique region (VP1u) from the viral capsid, which mediates receptor engagement. Our prior studies identified AXL as a co-receptor for B19V infection of EPCs. Here, we identify that human transferrin receptor 1 (hTfR) as a critical co-receptor that directly binds VP1u and promotes viral internalization. Inhibition of the VP1u-hTfR interaction by competitive binding of hTfR with either an anti-hTfR monoclonal antibody or human ferritin significantly reduces B19V internalization and replication in ex vivo -expanded EPCs, highlighting a link between VP1u binding to the apical domain of hTfR and viral internalization. RBD mutants that disrupt its interaction with hTfR barely inhibited B19V infection in EPCs. These findings support a model in which hTfR directly engages VP1u and promotes B19V internalization into EPCs. Defining these mechanisms provides a foundation for developing antiviral strategies targeting B19V entry into EPCs.