Graham, S. V. | Search bioRxiv

bioRxiv2025

Human Rhinovirus 16 impacts cilia structure in 3D cultured primary bronchial epithelial tissue through alternative splicing of host cilia RNAs.

Human rhinoviruses (HRV) are a leading cause of the common cold but can often lead to respiratory complications such as wheeze in young children. In a transcriptomic study of respiratory nasal swab specimens from children hospitalised with acute wheeze, a significant alteration was found in the expression of the serine/arginine rich splicing factor (SRSF) kinase, SRPK1, between HRV positive children with acute exacerbations and HRV negative controls. As this kinase can regulate host RNA splicing, we hypothesised that HRV infection could dysregulate the expression of host mRNAs to affect antiviral functions or to alter the morphological features of the infected respiratory epithelium. Here, we show that pharmacological inhibition of SRPK1 in primary bronchial epithelial cells resulted in increased HRV16 replication while overexpression of SRPK1 reduced viral replication. In a primary bronchial epithelial 3D model infected with HRV16 decreased phosphorylation of SRSF1, 3 and 6 was observed. Furthermore, transcriptomic and alternative splicing (AS) bioinformatic analysis revealed the significantly altered AS of 1228 host genes during infection. Subsequent pathway analysis revealed the enrichment of most of these genes in networks related to cilia development and function. HRV16 infection led to significantly decreased cilia length and total cilia numbers in the primary bronchial epithelial 3D model together with changes to selected cilia proteins. Overall, this investigation has unravelled novel cellular networks implemented during HRV infection that may lead to acute exacerbations of respiratory infections. Author summaryHuman rhinoviruses cause the common cold. In immunocompetent individuals this is usually a self-limiting infection. However, in young children and the elderly, infection can lead to complications such as bronchiolitis, croup, and wheezing. Rhinovirus infection can exacerbate chronic conditions such as cystic fibrosis, chronic obstructive pulmonary disease and asthma. Understanding the molecular pathology of this exacerbation could lead to new avenues for therapy. In this study, we discovered that a multifunctional cellular enzyme called serine arginine protein kinase 1 (SRPK1) is a restriction factor for human rhinovirus 16 (HRV16) infection. One key cellular function of SRPK1 is to regulate RNA splicing through modifying the SR proteins that normally enhance splicing. In three dimensional tissues grown from human bronchial epithelial cells, we found that HRV16 infection led to decreased levels of modified SR proteins. This change resulted in significant alterations in RNA expression in the infected cell. Most of these alterations affected production of the correct versions of cilia proteins resulting in reduced cilia numbers and cilia blunting. This type of damage due to HRV infection would result in inefficient clearance of subsequent viral infections prolonging the viral infection leading to lower respiratory tract infection and to exacerbations of existing chronic disease.

microbiology↗

bioRxiv2016

Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

The herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.\n\nAuthor SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

cell biology↗