Characterization of Human Immunodeficiency Virus-1 Transmission Clusters and Transmitted Drug-Resistant Mutations in Croatia from 2019 to 2022.

Molecular epidemiology of HIV-1 infection is challenging due to the highly diverse HIV-genome. We investigated the genetic diversity and prevalence of transmitted drug resistance (TDR) followed by phylogenetic analysis in 270 HIV-1 infected, treatment-naïve individuals from Croatia in the period 2019-2022. The results of this research confirmed a high overall prevalence of TDR of 16.7%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTIs (NNRTIs), and protease inhibitors (PIs) was found in 9.6%, 7.4%, and 1.5% of persons, respectively. No resistance to integrase strand-transfer inhibitors (INSTIs) was found. Phylogenetic analysis revealed that 173/229 sequences (75.5%) were part of transmission clusters, and the largest identified was T215S, consisting of 45 sequences. Forward transmission was confirmed in several clusters. We compared deep sequencing (DS) with Sanger sequencing (SS) on 60 randomly selected samples and identified additional surveillance drug resistance mutations (SDRMs) in 49 of them. Our data highlight the need for baseline resistance testing in treatment-naïve persons. Although no major INSTIs were found, monitoring of SDRMs to INSTIs should be continued due to the extensive use of first- and second-generation INSTIs.

Effects of S-Adenosylhomocysteine Hydrolase Downregulation on Wnt Signaling Pathway in SW480 Cells.

S-adenosylhomocysteine hydrolase (AHCY) deficiency results mainly in hypermethioninemia, developmental delay, and is potentially fatal. In order to shed new light on molecular aspects of AHCY deficiency, in particular any changes at transcriptome level, we enabled knockdown of AHCY expression in the colon cancer cell line SW480 to simulate the environment occurring in AHCY deficient individuals. The SW480 cell line is well known for elevated AHCY expression, and thereby represents a suitable model system, in particular as AHCY expression is regulated by MYC, which, on the other hand, is involved in Wnt signaling and the regulation of Wnt-related genes, such as the ß-catenin co-transcription factor LEF1 (lymphoid enhancer-binding factor 1). We selected LEF1 as a potential target to investigate its association with S-adenosylhomocysteine hydrolase deficiency. This decision was prompted by our analysis of RNA-Seq data, which revealed significant changes in the expression of genes related to the Wnt signaling pathway and genes involved in processes responsible for epithelial-mesenchymal transition (EMT) and cell proliferation. Notably, LEF1 emerged as a common factor in these processes, showing increased expression both on mRNA and protein levels. Additionally, we show alterations in interconnected signaling pathways linked to LEF1, causing gene expression changes with broad effects on cell cycle regulation, tumor microenvironment, and implications to cell invasion and metastasis. In summary, we provide a new link between AHCY deficiency and LEF1 serving as a mediator of changes to the Wnt signaling pathway, thereby indicating potential connections of AHCY expression and cancer cell phenotype, as Wnt signaling is frequently associated with cancer development, including colorectal cancer (CRC).

HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia.

IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

Long-Term Culturing of FreeStyle 293-F Cells Affects Immunoglobulin G Glycome Composition.

Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell "aging". To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG.

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1.

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.

Diverse SARS-CoV-2 variants preceded the initial COVID-19 outbreak in Croatia.

We developed a next-generation SARS-CoV-2 sequencing platform and obtained the first SARS-CoV-2 sequences from patients in Croatia at the beginning of the COVID-19 outbreak in the spring of 2020. Integrating the sequencing and the epidemiological data, we show that patients were infected with different SARS-CoV-2 variants belonging to different clades (mostly G and GH). This result confirms that there was widespread virus transmission early in 2020. Interestingly, we identified a unique mutation resulting in a V13I substitution in Nsp5A, the main viral protease, in a patient who had not received antiviral therapy.

Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines.

Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics.

Lowe syndrome - Old and new evidence of secondary mitochondrial dysfunction.

The oculocerebrorenal syndrome of Lowe (LS) is a rare, progressive, multisystemic X-linked disorder caused by mutations in OCRL gene. Patients classically present with ocular abnormalities including bilateral congenital cataracts and glaucoma, intellectual delay, severe generalized hypotonia with absent tendon reflexes, and proximal renal tubular dysfunction. Congenital bilateral cataracts and hypotonia are present at birth in almost all patients, while other classical symptoms develop gradually with variable severity. Consequently, differential diagnosis in infant period in these patients can be broad including other rare metabolic and neurologic disorders. Herein we present a 4.5 year old boy with Lowe syndrome caused by mutation of OCRL gene, NM_000276.4:c.643C > T; p.(Gln215*), initially diagnosed as having mitochondriopathy due to alteration of mitochondria on electron microscopic examination in different tissues and decreased values of mitochondrial energy metabolism measurements in muscle. No pathogenic mutations in mitochondrial DNA were found on whole exome sequencing. This patient recall historical hypothesis of secondary mitochondrial dysfunction in Lowe syndrome, that may be caused/intensified by some of disease symptoms.

Analysis of HIV-1 diversity, primary drug resistance and transmission networks in Croatia.

Molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia was investigated. We included 403 persons, representing 92.4% of all HIV-positive individuals entering clinical care in Croatia in 2014-2017. Overall prevalence of transmitted drug resistance (TDR) was estimated at 16.4%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTI (NNRTIs) and protease inhibitors (PIs) was found in 11.4%, 6.7% and 2.5% of persons, respectively. Triple-class resistance was determined in 2.2% of individuals. In addition, a single case (1.0%) of resistance to integrase strand-transfer inhibitors (InSTIs) was found. Deep sequencing was performed on 48 randomly selected samples and detected additional TDR mutations in 6 cases. Phylogenetic inference showed that 347/403 sequences (86.1%) were part of transmission clusters and identified forward transmission of resistance in Croatia, even that of triple-class resistance. The largest TDR cluster of 53 persons with T215S was estimated to originate in the year 1992. Our data show a continuing need for pre-treatment HIV resistance testing in Croatia. Even though a low prevalence of resistance to InSTI was observed, surveillance of TDR to InSTI should be continued.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

Herpes simplex virus 1 miRNA sequence variations in latently infected human trigeminal ganglia.

Human herpes simplex virus 1 (HSV-1) expresses numerous miRNAs, the function of which is not well understood. Several qualitative and quantitative analyses of HSV-1 miRNAs have been performed on infected cells in culture and animal models, however, there is very limited knowledge of their expression in human samples. We sequenced small-RNA libraries of RNA derived from human trigeminal ganglia latently infected with HSV-1 and Varicella zoster virus (VZV) and detected only a small subset of HSV-1 miRNA. The most abundantly expressed miRNAs are miR-H2, miRNA that regulates the expression of immediate early gene ICP0, and miR-H3 and -H4, both miRNAs expressed antisense to the transcript encoding the major neurovirulence factor ICP34.5. The sequence of many HSV-1 miRNAs detected in human samples was different from the sequences deposited in miRBase, which might significantly affect targeted functional analyses.

Mutations in S-adenosylhomocysteine hydrolase (AHCY) affect its nucleocytoplasmic distribution and capability to interact with S-adenosylhomocysteine hydrolase-like 1 protein.

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.