The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles

Abstract Background The process of HIV-1 genomic RNA (gRNA) encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ). Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site) from the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV) vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles. Results We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ). Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form. Conclusions Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation

Insights into the mechanisms of HIV-1 cis elements and trans factors required for RNA encapsidation and transduction

The retroviral replication process is typically separated into early events of infection for the virus to enter a host cell, and late events that generate new viral particles. Encapsidation (late event) and early infection events are governed by several different cis elements located in the viral RNA, and viral, or cellular, trans factors (proteins). A number of viral encoded components have been identified to function in the encapsidation process, most notably the Gag polyprotein and genomic RNA cis elements in the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is also a burgeoning role for the Rev/RRE in the encapsidation process. Additionally, cis elements of the RNAs encapsidated into viral particles may influence different early stages of infection into host cells. This dissertation employs an innovative approach that affords separation of cis and trans viral components to investigate their independent, and combined, effects on encapsidation and early events of infection that will be referred to as transduction. HIV-1 cis elements were reconstructed in the context of heterologous RNAs to assess encapsidation and transduction functions. This work demonstrates for the first time that the Rev/RRE system can augment heterologous RNA encapsidation independent of all cis elements from the 5' UTR. In fact, the Rev/RRE system appears to be required for specific and efficient encapsidation into HIV-1 viral particles, a process more commonly associated with Gag recognition of the canonical packaging signal in the 5' UTR. Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to ensuing interactions between Gag and the canonical packaging signal for subsequent encapsidation. Lastly, we show that heterologous RNAs can conform to transduction properties commonly associated with HIV-1 viral particles. Furthermore, some heterologous RNAs exhibit an episomal profile in transduced cells that may have improved safety benefits over more conventional nonintegrating lentiviral vectors. These innovative vector systems may prove beneficial for therapeutic gene delivery to nondividing cells

An effective DNA vaccine platform for Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) is an ongoing emerging infectious disease across the Arabian Peninsula, with the majority of cases occurring in Saudi Arabia. Through September 23, 2016 the World Health Organization reported about 1,806 total cases, including 643 deaths from 27 countries (http://www.who.int/emergencies/mers-cov/en/). The disease is comprised of a lower respiratory infection wherein individuals exhibit pneumonia-like symptoms that often lead to multi-organ failure and death (1). In addition to close contact with infected camels, transmission from human-to-human most commonly occurs in the hospital setting through close contact between patients and hospital workers (1). MERS-CoV has also been isolated from objects within patient rooms including bed sheets, bed rails, and IV fluid hangers (2), which may all be potential sources of transmission. Several cases of MERS-CoV have been associated with travelers returning home from the Middle East and developing symptoms, including two cases of health care workers returning to the United States (3). The potential for global spread was recently illustrated by a South Korean national returning home from visiting the Arabian Peninsula in May, 2015, and initiating an outbreak that infected 186 people resulting in 20% mortality and a nationwide economic crisis (4). Nonetheless, MERS-CoV is not thought to be sustained in the human population through human-to-human transmission, but may instead be continuously re-introduced into the human population from a zoonotic source, most likely dromedary camels because of high seropositive rates in herds throughout the Middle East (5,6). As camels are integral to the Saudi Arabian culture and economy, nationwide culling of camel herds is not feasible. Consequently, camel vaccination is being considered (7); however, therapeutic strategies have primarily focused on interfering with MERS-CoV infection in humans (3,5)

Glycosylation of Mouse DPP4 Plays a Role in Inhibiting Middle East Respiratory Syndrome Coronavirus Infection

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development

Modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice

The emergence of highly pathogenic human coronaviruses (hCoVs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. Global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. Limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hCoV infections. Therefore, understanding how hCoV-host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hCoVs in humans. Pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hCoV-host interactions that govern pathogenesis. Notably, tractable mouse genetics combined with hCoV reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus-host interaction networks in disease. In addition to assessing etiologies of known hCoVs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent CoVs. Knowledge of CoV pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the World Health Organization. Although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hCoV mouse models, their use as tractable complex genetic organisms for untangling complex hCoV-host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions

Mouse Dipeptidyl Peptidase 4 Is Not a Functional Receptor for Middle East Respiratory Syndrome Coronavirus Infection

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics

Adaptive evolution influences the infectious dose of MERS-CoV necessary to achieve severe respiratory disease

We recently established a mouse model (288–330+/+) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 106 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 103 and 105 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10–1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms, and multi-organ failure, with a case fatality rate of ~36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibited pathology consistent with late stages of ARDS, which is reminiscent of disease observed in patients infected with SARS coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea pig, and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR/Cas9 to modify the mouse genome to encode two human amino acids (288 and 330) in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs, and evoked symptoms indicative of severe acute respiratory distress syndrome (ARDS), including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary hemorrhage, and pathological signs indicative of end stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected engineered mice against MERS-CoV-induced ARDS

MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis

ABSTRACT While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants

X-Linked Inhibitor of Apoptosis Protein-Mediated Attenuation of Apoptosis, Using a Novel Cardiac-Enhanced Adeno-Associated Viral Vector

Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector