Biology subjects

Akbar, H.

Publications and source records attributed to Akbar, H..

4 recordsLinked to original sources

bioRxiv2026

Temporal phase-resolved transcriptomics reveals host determinants of Mareks disease virus reactivation in transformed chicken T cells

Mareks disease virus (MDV) is an oncogenic herpesvirus that establishes latency in CD4+ T cells, from which it reactivates to initiate productive replication and dissemination. To define host mechanisms governing the transition from latency to early and late lytic replication, we developed recombinant MDV expressing early RLORF4mRFP and late UL47eGFP, which was used to isolate latent (Lo; mRFP low), early lytic (Hi; mRFP high), and late lytic (DP; mRFP+eGFP+) populations from MDV-induced lymphoblastoid cell lines (LCLs). Two independent LCLs (Lines 62 and 82) were subjected to Illumina RNA sequencing after sorting-purified populations. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, whereas Line 62 showed only two differentially expressed viral genes. However, both lines converged on a highly conserved transcriptional program during the transition from latency (Lo) to late-lytic replication (DP), sharing 118 viral transcripts, most of which were structural and assembly-associated genes. Among the host transcriptional responses, Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, and RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic, and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which the cellular state governs the initiation of the lytic switch, whereas downstream replication proceeds through a conserved viral program.

microbiology↗

bioRxiv2026

Conserved herpesvirus protein kinase (CHPK)-mediated phosphorylation of viral proteins associated with nucleocytoplasmic trafficking during natural infection

The conserved herpesvirus protein kinase (CHPK) is encoded by all members of the Orthoherpesviridae and contributes to replication in cell culture but is not strictly required. Mareks disease virus (MDV) CHPK is dispensable for replication in cultured cells yet essential for horizontal transmission in chickens. To elucidate its role during natural infection, we performed RNA sequencing (RNA-seq) and mass spectrometry (MS)-based phosphoproteomics on spleen and feather follicle epithelial skin cells from chickens infected with wild-type or CHPK-null MDV. RNA-seq detected only a limited number of viral transcripts in the spleen--including latency-associated transcripts (LATs) and the major oncogene Meq--with minimal differences between wild-type and CHPK-null infections. In feather follicle epithelial skin cells, the full repertoire of viral genes was expressed, but only seven genes showed differential expression between wild-type and CHPK-null viruses. In striking contrast, MS-based phosphoproteomics identified many differentially phosphorylated proteins, including 21 viral proteins. These findings indicate that CHPKs critical functions in skin replication and subsequent horizontal transmission are primarily mediated through post-translational modifications (PTMs) rather than transcriptional regulation. Among the CHPK-targeted viral proteins were three MDV-unique proteins, eight conserved within the Alphaherpesvirinae, and ten conserved across the Orthoherpesviridae. In silico analysis revealed that many differentially phosphorylated serine and threonine residues lie near or within predicted nuclear localization signals (NLS) and nuclear export signals (NES). Functional validation confirmed that several of these motifs actively control nucleocytoplasmic shuttling of the respective viral proteins. Collectively, these data suggest that MDV CHPK orchestrates the subcellular localization of multiple viral proteins in epithelial skin cells via phosphorylation, thereby enabling efficient replication and horizontal transmission in the natural host. AUTHOR SUMMARYUnderstanding the mechanisms by which herpesviruses replicate and spread within their natural hosts and identifying the viral genes essential for these processes are fundamental to developing effective antiviral strategies. Mareks disease virus (MDV), a highly contagious alphaherpesvirus, remains a major economic threat to the global poultry industry while serving as a powerful natural animal model for studying herpesvirus pathogenesis and transmission in vivo. Using an established in vivo enrichment method for infected cells, we conducted a comprehensive analysis of viral gene expression, protein abundance, and post-translational modifications (PTMs) during natural infection. Remarkably, RNA sequencing revealed virtually no differences in viral transcription between wild-type and CHPK-null viruses in either spleen or feather follicle epithelial skin cells. In contrast, phosphoproteomics showed that CHPK extensively regulates the phosphorylation of multiple viral proteins specifically in skin epithelial cells. In silico and functional analyses further indicate that these CHPK-mediated phosphorylations occur near or within nuclear localization (NLS) and nuclear export (NES) signals, directly controlling the nucleocytoplasmic shuttling of key viral proteins. This work suggests CHPK as a master regulator of viral protein subcellular localization during replication in the natural host and highlights CHPK orthologs as promising broad-spectrum therapeutic targets against herpesviruses.

microbiology↗

bioRxiv2023

Viral proteogenomic and expression profiling during fully productive replication of a skin-tropic herpesvirus in the natural host

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to their close relationship to their natural host. Mareks disease is a devastating herpesviral disease of chickens caused by Mareks disease virus (MDV) and an excellent natural model to study skin- tropic herpesviruses and transmission. Similar to varicella zoster virus that causes chicken pox in humans, the only site where fully productive replication occurs is in epithelial skin cells and this is required for host to host transmission. Here, we enriched for actively replicating virus in feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via both intron-spanning sequencing reads as well as a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5 ends of two core herpesviral genes, pUL47 and ICP4, along with evidence of transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems. Author SummaryIn the natural host, the transcriptome and proteome of many herpesviruses are poorly defined. Here, we evaluated the viral transcriptome and proteome in feather follicle epithelial skin cells of chickens infected with Mareks disease virus (MDV), an important poultry pathogen as well as an excellent model for skin-tropic human alphaherpesvirus replication in skin cells. Using fluorescently tagged virus, we significantly enriched the number of infected cells sampled from live chickens, greatly enhancing the detection of viral transcripts and proteins within a host background. Based on this, we could confirm the translation of most transcripts using deep MS/MS-based proteomics and identify novel expressed peptides supportive of an increasingly complex translational and regulatory viral landscape. The demonstrated deep peptide sequencing capability can serve as a template for future work in herpesviral proteomics.

microbiology↗

bioRxiv2022

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK)

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Mareks disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK--particularly its kinase activity--is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection. Author SummaryMareks disease herpesvirus (MDV) is an important pathogen in the poultry industry. Current vaccines reduce disease but do not protect chickens from infection. Understanding natural infection provides vital information for developing potential therapies to protect against Mareks disease, while also providing a robust natural virus-host model to study herpesvirus pathogenesis. The conserved Herpesviridae protein kinase (CHPK) of MDV is essential for the initiation of natural infection, and we identified the virion protein US10 as a potential target for CHPK during natural infection. Our results showed that MDV CHPK was required for expression of pUS10 in both cell culture and in chickens. Although MDV lacking pUS10 replicated and spread in experimentally infected chickens, it was less virulent implicating an important role of pUS10 in natural infection of the host. These results emphasize the importance of studying specific viral proteins and their functions during infection of the natural host.

microbiology↗