Fusion of Large Polypeptides to Human Adenovirus Type 5 Capsid Protein IX Can Compromise Virion Stability and DNA Packaging Capacity.

The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function.IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.

Human adenoviral DNA association with nucleosomes containing histone variant H3.3 during the early phase of infection is not dependent on viral transcription or replication.

Adenovirus (Ad) DNA undergoes dynamic changes in protein association as the virus progresses through its replicative cycle. Within the virion, the Ad DNA associates primarily with the virus-encoded, protamine-like protein VII. During the early phase of infection (∼6 h), the viral DNA showed declining association with VII, suggesting that VII was removed from at least some regions of the viral DNA. Within 6 h, the viral DNA was wrapped into a repeating nucleosome-like array containing the histone variant H3.3. Transcription elongation was not required to strip VII from the viral DNA or for deposition of H3.3. H3.1 did not associate with the viral DNA at any point during infection. During the late phase of infection (i.e., active DNA replication ∼12-24 h), association with H3 was dramatically reduced and the repeating nucleosome-like pattern was no longer evident. Thus, we have uncovered some of the changes in nucleoprotein structure that occur during lytic Ad infection.

Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

Assembly of helper-dependent adenovirus DNA into chromatin promotes efficient gene expression.

Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2- to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

Rational design of murine secreted alkaline phosphatase for enhanced performance as a reporter gene in mouse gene therapy preclinical studies.

Many preclinical gene therapy studies use a reporter gene to evaluate vector design and performance in mouse models of human disease. Unfortunately, most commonly used reporter genes are immunogenic in mice, which confounds accurate evaluation of vector function. In previous studies, we showed that the murine secreted alkaline phosphatase (mSEAP) gene functions well as a simple and sensitive reporter gene in mice. In this study, we have used rational design to enhance mSEAP performance. The majority of native mSEAP remains attached to the outer surface of the cell through glycan phosphatidylinositol linkage; removal of the carboxy-terminal tail of mSEAP resulted in a dramatic enhancement of release of the protein into cell culture medium and into mouse plasma in whole animal experiments. We increased the heat stability of mSEAP through mutation of a key residue in the crown domain of the protein (H451E), thus allowing us to reduce endogenous, background AP activity through heat inactivation for enhanced sensitivity. We show that these alterations in mSEAP result in enhanced performance in tissue culture and mouse studies. Taken together, these data illustrate that mSEAP is a sensitive, nonimmunogenic reporter for preclinical mouse studies.

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.