Biology subjects

Geballe, A. P.

Publications and source records attributed to Geballe, A. P..

7 recordsLinked to original sources

bioRxiv2025

Replication-Deficient Whole-Virus Vaccines Against Cytomegalovirus Induce Protective Immunity in a Guinea Pig Congenital Infection Model

Vaccines are needed to prevent congenital cytomegalovirus (CMV) infections. This study used the guinea pig cytomegalovirus (GPCMV) model to examine replication-deficient whole-virus vaccines against maternal viremia and congenital infection. Two recombinant GPCMVs, GP51-DD and GP52-DD, were engineered with destabilization domains fused to essential viral late proteins GP51 and GP52. These viruses, predicted to replicate in the presence of the synthetic ligand Shield-1 but not in its absence, were evaluated for Shield-1-dependence in vitro and for safety, immunogenicity, and efficacy in a pregnancy/challenge model. GP52-DD was profoundly Shield-1-dependent, producing no detectable infectious progeny in its absence. In contrast, the replication of GP51-DD was delayed in the absence of Shield-1, but the virus reached similar peak titers with or without the compound. GPCMV-seronegative guinea pigs received two subcutaneous injections of placebo (sham-immunized), GP51-DD, GP52-DD, or wild-type GPCMV (WT-GPCMV). DNAemia post-vaccination was detected in only one of 14 dams in each of the GP51-DD- and GP52-DD-immunized groups compared to 10/10 control animals immunized with WT-GPCMV, confirming the recombinant viruses were attenuated. GPCMV-specific antibody responses were similar in all three vaccinated groups. When immunized dams were bred and challenged with virulent GPCMV, DNAemia was detected in all sham-immunized controls and in 44% of GP52-DD-immunized dams (at significantly reduced levels) but was absent in dams immunized with GP51-DD or WT-GPCMV. Notably, immunization with GP52-DD, GP51-DD, or WT-GPCMV significantly reduced congenital GPCMV transmission (protective efficacies of 89%, 94%, and 100%, respectively). Thus, the replication-deficient GP52-DD vaccine was attenuated and protected against intrauterine GPCMV transmission. ImportanceCongenital cytomegalovirus (CMV) infections could be prevented by a vaccine, but most of the vaccine designs that have advanced to clinical trials have been subunit vaccines. These viral envelope glycoprotein-targeting designs have not yielded an effective vaccine that provides durable immunity. A vaccine that confers immune responses to a broader repertoire of viral immunogens, such as vaccines based on the whole virus, could enhance protection. However, there are concerns about the safety of live attenuated CMV vaccines. Using the guinea pig/guinea pig cytomegalovirus model of congenital infection, this study demonstrates that two replication-deficient whole virus vaccines are attenuated in animals while also being highly immunogenic, providing protective immunity against maternal viremia and fetal infection at levels similar to that conferred by a wild-type virus.

immunology↗

bioRxiv2024

Human cytomegalovirus induces neuronal gene expression for viral maturation

Viral invasion of the host cell causes some of the most dramatic changes in biology. Human cytomegalovirus (HCMV) extensively remodels host cells, altering nuclear shape and generating a cytoplasmic viral-induced assembly compartment (vIAC). How these striking morphology changes take place in the context of host gene regulation is still emerging. Here, we discovered that histone variant macroH2A1 is essential for producing infectious progeny. Because virion maturation and cellular remodeling are closely linked processes, we investigated structural changes in the host cell upon HCMV infection. We discovered that macroH2A1 is necessary for HCMV-induced reorganization of the host nucleus, cytoskeleton, and endoplasmic reticulum. Furthermore, using RNA-seq we found that while all viral genes were highly expressed in the absence of macroH2A1, many HCMV-induced host genes were not. Remarkably, hundreds of these HCMV-induced macroH2A1-dependent host genes are associated with neuronal synapse formation and vesicle trafficking. Knock-down of these HCMV-induced neuronal genes during infection resulted in malformed vIACs and smaller plaques, establishing their importance to HCMV infection. Together, our findings demonstrate that HCMV manipulates host gene expression by hijacking a dormant neuronal secretory pathway for efficient virion maturation.

microbiology↗

bioRxiv2023

A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB

Myxovirus resistance proteins (MxA and MxB) are interferon-induced proteins that exert antiviral activity against a diverse range of RNA and DNA viruses. In primates, MxA has been shown to inhibit myxoviruses, bunyaviruses, and hepatitis B virus, whereas MxB restricts retroviruses and herpesviruses. As a result of their conflicts with viruses, both genes have been undergoing diversifying selection during primate evolution. Here, we investigate how MxB evolution in primates has affected its restriction of herpesviruses. In contrast to human MxB, we find that most primate orthologs, including the closely related chimpanzee MxB, do not inhibit HSV-1 replication. However, all primate MxB orthologs tested restrict human cytomegalovirus. Through the generation of human and chimpanzee MxB chimeras we show that a single residue, M83, is the key determinant of restriction of HSV-1 replication. Humans are the only primate species known to encode a methionine at this position, whereas most other primate species encode a lysine. Residue 83 is also the most polymorphic residue in MxB in human populations, with M83 being the most common variant. However, [~]2.5% of human MxB alleles encode a threonine at this position, which does not restrict HSV-1. Thus, a single amino acid variant in MxB, which has recently risen to high frequency in humans, has endowed humans with HSV-1 antiviral activity. ImportanceHerpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections as well as how viruses can evolve to counteract these host defenses is critically important for understanding viral disease pathogenesis, and for developing therapeutic tools aimed at treating or preventing viral infections. Additionally, understanding how these host and viral mechanisms adapt to counter one another can aid in identifying the risks of, and barriers to, cross-species transmission events. As highlighted by the recent SARS-CoV-2 pandemic, episodic transmission events can have severe consequences for human health. This study reveals that the major human variant of the antiviral protein MxB inhibits the human pathogen HSV-1, whereas human minor variants and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the defense systems of their native hosts, in this case the human gene appears to be at least temporarily winning at this interface of the primate-herpesviral evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxBs ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

microbiology↗

bioRxiv2023

Polypharmacology-based kinome screen identifies new regulators of KSHV reactivation

Kaposis sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposis sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) causes Kaposis sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

microbiology↗

bioRxiv2022

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived PKR antagonist RhTRS1 in place of its native PKR antagonists; E3L and K3L (VACV{Delta}E{Delta}K+RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a "molecular foothold" to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACV{Delta}E{Delta}K+RhTRS1 replication in human cells, mediated by both PKR and RNase L. We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage nine. Using our Illumina-based pipeline, we found that some SNPs which had evolved during the prior AGM adaptation were rapidly lost, while 13 singlebase substitutions and short indels increased over time, including two SNPs unique to HFF adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an "intermediate species" and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

evolutionary biology↗

bioRxiv2022

Translational buffering by ribosome stalling in upstream open reading frames

Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames by several mechanisms: siphoning away scanning ribo-somes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5 UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF translation when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF translation via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.

molecular biology↗

bioRxiv2020

Rapid adaptation to human protein kinase R by a unique genomic rearrangement in rhesus cytomegalovirus.

Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved fitness in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles. AUTHOR SUMMARYRhesus macaque CMV (RhCMV) is an important model for human CMV (HCMV) pathogenesis and vaccine development. Therefore, it is important to understand the similarities and differences in infectivity and interaction of these viruses with their host species. In contrast to the strict species-specificity of HCMV, RhCMV is able to cross species barriers to replicate in human cells. We know from past work that a component of this broader host range is RhCMVs ability to counteract both the rhesus and human versions of a key antiviral factor. Here we delve further into the mechanisms by which RhCMV can adapt to counteract human cellular defenses. We find that RhCMV appears to be poised to undergo a specific genomic rearrangement that facilitates increased replication efficiency in human cells. Besides providing insights into CMV species-specificity and host barriers to cross-species transmission, this work also provides more generalized clues about viral adaptative mechanisms.

microbiology↗