Biology subjects

Krishna, B. A.

Publications and source records attributed to Krishna, B. A..

7 recordsLinked to original sources

bioRxiv2025

Viral Diversity Influences T Cell Responses to Enteric Human Adenoviruses F40 and F41

BackgroundHuman enteric species F adenoviruses are a leading cause of diarrhoea-associated paediatric morbidity and mortality worldwide. The cellular immune response (antigen-specific cytotoxic T cells and secreted cytokines) to human adenovirus (HAdV) infection is known to ameliorate symptoms and is critical for viral clearance. We hypothesised that the capsid proteins (hexon and penton) of HAdV-F40 and 41 (F40, F41) are evolving to escape cellular immune responses. Major histocompatibility complex (MHC) binding of viral peptides is a key step in the presentation of peptide-MHC complexes which activate the T cell receptor and the cytotoxic T cell response. MethodsUsing global HAdV genomic data, we predicted MHC-peptide binding within the hexon and penton proteins of F40 and F41. We focused on MHC class I alleles common in the UK and Kenya and identifying predicted CD8+ T cell epitopes. Eight predicted epitope pairs from the F41 hexon were synthesised as 15mer peptides, comparing the wildtype (1970 F41 reference) to the variant (2019-2022) sequences. Cellular IFN{gamma} responses to these epitopes were measured in healthy donors using FluoroSpot assays. ResultsWe identified multiple predicted CD8+ T cell epitopes shared between HAdV-species C and F, but also unique to species F, and epitopes unique to each genotype. We show that IFN{gamma} and IL2 peripheral blood mononuclear cell (PBMC) responses to HAdV-F are ubiquitous among healthy adult donors from Cambridge, UK. Among predicted CD8+ epitopes within the F41 hexon, 11/16 peptides elicited donor positive IFN{gamma} responses from healthy donor PBMC (at least one epitope from seven out of eight peptide pairs). ConclusionsThe hexon and penton proteins of HAdV-F-40 and F41 are predicted to contain a number of genotype-specific, but conserved, CD8+ T cell epitopes which could be used to inform future vaccine design. Using the hexon of F41 as a case study, we show that predicted T cell epitopes in emergent strains are able to elicit an inflammatory cytokine response from healthy donor PBMC. The role of T cell recognition in driving enteric adenovirus evolution deserves further consideration.

microbiology↗

bioRxiv2025

Human cytomegalovirus-encoded G protein-coupled receptor (GPCR), UL78, regulates viral reactivation

Human cytomegalovirus (CMV) is a ubiquitous pathogen that establishes life-long, latent infection in hematopoietic cells. Immune-competent individuals are usually asymptomatic for disease. However, immune dysregulation in latently-infected individuals can result in viral reactivation, often causing further complications. Viral gene transcription during latency is restricted, although the CMV-encoded G-protein coupled receptor homologs, US28 and UL78, are expressed. We and others find US28 is critical for establishing and maintaining viral latency, in part, through regulating host cell signaling. How US28 switches from pro-latent to pro-lytic during reactivation, however, is unknown, though our findings herein reveal a role for UL78. Myeloid cells infected with a UL78 ORF deletion mutant maintain viral latency yet fail to efficiently reactivate. However, the UL78 G protein-coupling domain is not required for reactivation, suggesting UL78-mediated signaling is not critical for reactivation. Prior work revealed UL78 and US28 interact, resulting in altered US28-mediated signaling. Additionally, we showed US28 attenuates ERK phosphorylation during latency, while ERK is phosphorylated upon reactivation; however, the mechanism underlying this switch is unknown. Thus, we hypothesized the UL78:US28 interaction is important for altering US28-mediating signaling upon viral reactivation. We find US28 and UL78 interact during lytic infection of fibroblasts and colocalize in myeloid cells upon their differentiation. Further, reactivation in myeloid cells latently-infected with wild-type virus results in upregulated ERK phosphorylation, while parallel cultures infected with the UL78-deficient virus fail to do so. Our data reveal the first function for UL78 in myeloid cells, where it influences cellular signaling to switch from pro-latent to pro-lytic. ImportanceCytomegalovirus (CMV) is a ubiquitous human herpesvirus, infecting the majority of the population worldwide. As with all herpesviruses, once an individual is infected with CMV, the virus remains in a persons blood cells for their life in a silenced state called latency, and this infection, for the most part, remains asymptomatic. When an infected individuals immune system fails to function properly, however, CMV can become active (termed viral reactivation), which allows the virus to replicate and cause downstream disease. Our understanding of the cellular and viral factors that dictate this switch from silenced to activated remains incomplete. Here, we show a viral protein, UL78, is required for this switch. We find UL78 helps to reshape cellular signaling, changing the cell environment from one that favors latency to one that instead supports reactivation. This highlights a new avenue for therapeutic intervention to prevent CMV reactivation and downstream disease.

microbiology↗

bioRxiv2025

Inhibition of MAPK signaling suppresses cytomegalovirus reactivation in CD34+ Kasumi-3 cells

Reactivation of latent human cytomegalovirus (CMV) can lead to severe complications in individuals with dysregulated immune systems. While antiviral therapies for CMV are approved, these compounds are limited by their toxicity and inability to specifically target the latent reservoir or prevent reactivation. Herein we show that CMV reactivation in Kasumi-3 cells, a CD34+ hematopoietic cell line, requires mitogen-activated protein kinase (MAPK) activation. Importantly, pharmacological inhibition of the MAPK signaling pathway, including MEK and ERK, restricts viral reactivation in Kasumi-3 cells. In sum, our findings show MAPK signaling is critical for CMV reactivation, revealing a potential avenue for therapeutic intervention to prevent viral reactivation and downstream pathogenesis that is often detrimental for immunosuppressed and immunocompromised patients.

microbiology↗

bioRxiv2020

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency. Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.

microbiology↗

bioRxiv2020

The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

microbiology↗

bioRxiv2019

Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimise latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that MHC Class II and two PYHIN proteins, MNDA and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner, but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-{kappa}B, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.\n\nImportanceHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50-100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals, but is a serious cause of mortality and morbidity in the immunocompromised and in neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.

microbiology↗

bioRxiv2018

The human cytomegalovirus G-protein coupled receptor US28 promotes latency by attenuating c-fos

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, though the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G-protein coupled receptor (GPCR) homolog, US28 is required for successful latent infection. We now show that US28 protein (pUS28) provided in trans complements the US28{Delta} lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 hours. However, this repression is only maintained in the presence of continual pUS28 expression provided in trans. Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this, we find that US28{Delta} infection results in an increase in AP-1 binding to the MIEP, compared to wild type latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28{Delta} infection to similar levels we observe during wild type latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.\n\nSignificance StatementHuman cytomegalovirus (HCMV) is a wise-spread pathogen that remains with an individual for life in a quiescent/latent state, posing little threat to an otherwise healthy person. However, when an individuals immune system is severely compromised, HCMV can reactivate to its active/lytic state, resulting in viral spread and disease that is often fatal. The biological mechanisms underlying HCMV latency and reactivation remain poorly understood. Herein we show that the viral-encoded G-protein coupled receptor (GPCR) US28 aids in the establishment and the maintenance of viral latency. Furthermore, we find that US28 modulates host cell proteins to suppress viral processes associated with active/lytic replication, thereby promoting latent infection. This work provides mechanism by which HCMV modulates the host cell environment to its advantage.

microbiology↗