Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

Human immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 µm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11-12 µm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.

Screening for gene expression fluctuations reveals latency-promoting agents of HIV.

Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or "noise") that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration-approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.

Cell size dependent migration of T-cells latently infected with HIV.

Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent. In cell migration assays, migrating cells are consistently larger than non-migrating cells. This effect is drug-treatment independent. The cell size dependent motility observed in a previously generated Jurkat latency model correlates with the motility of primary human CD4+ T-cells containing a modified HIV-1 full-length construct JLatd2GFP. In addition, large migrating T-cells, latently infected with HIV, show a slightly decreased rate of reactivation from latency. these results demonstrate that HIV reactivation is cell migration-dependent, where host cell size acts as a catalyst for altered migration velocity. We believe that host cell size controlled migration uncovers an additional mechanism of cellular controlled viral fate determination important for virus dissemination and reactivation from latency. This observation may provide more insights into viral-host interactions regulating cell migration and reactivation from latency and helps in the design and implementation of novel therapeutic strategies.

Phenotypic and Genotypic Testing of HSV-1 and HSV-2 Resistance to Antivirals.

Resistance testing of antivirals to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can be done by phenotypic and genotypic methods. The determination of a resistant phenotype is based on the calculation of inhibitory concentrations for the antiviral drug, which should be tested. The main advantage of this resistance test is a clear interpretation of laboratory findings, but the method is time-consuming and a considerable experience is required by handling infectious virus. Genotypic resistance testing is based on the detection of resistance-related mutations in viral genes encoding the thymidine kinase and DNA polymerase, which need to be amplified and sequenced. This approach has the advantage of being faster, but only frameshift mutations, stops of translation, and amino acid substitutions described in the literature can be interpreted without doubt. By contrast, numerous novel amino acid substitutions are diagnostically less conclusive.

Cell Size-Based Decision-Making of a Viral Gene Circuit.

Latently infected T cells able to reinitiate viral propagation throughout the body remain a major barrier to curing HIV. Distinguishing between latently infected cells and uninfected cells will advance efforts for viral eradication. HIV decision-making between latency and active replication is stochastic, and drug cocktails that increase bursts of viral gene expression enhance reactivation from latency. Here, we show that a larger host-cell size provides a natural cellular mechanism for enhancing burst size of viral expression and is necessary to destabilize the latent state and bias viral decision-making. Latently infected Jurkat and primary CD4+ T cells reactivate exclusively in larger activated cells, while smaller cells remain silent. In addition, reactivation is cell-cycle dependent and can be modulated with cell-cycle-arresting compounds. Cell size and cell-cycle dependent decision-making of viral circuits may guide stochastic design strategies and applications in synthetic biology and may provide important determinants to advance diagnostics and therapies.

Fine-tuning of noise in gene expression with nucleosome remodeling.

Engineering stochastic fluctuations of gene expression (or "noise") is integral to precisely bias cellular-fate decisions and statistical phenotypes in both single-cell and multi-cellular systems. Epigenetic regulation has been shown to constitute a large source of noise, and thus, engineering stochasticity is deeply intertwined with epigenetics. Here, utilizing chromatin remodeling, we report that Caffeic acid phenethyl ester (CA) and Pyrimethamine (PYR), two inhibitors of BAF250a, a subunit of the Brahma-associated factor (BAF) nucleosome remodeling complex, enable differential and tunable control of noise in transcription and translation from the human immunodeficiency virus long terminal repeat promoter in a dose and time-dependent manner. CA conserves noise levels while increasing mean abundance, resulting in direct tuning of the transcriptional burst size, while PYR strictly increases transcriptional initiation frequency while conserving a constant transcriptional burst size. Time-dependent treatment with CA reveals non-continuous tuning with noise oscillating at a constant mean abundance at early time points and the burst size increasing for treatments after 5 h. Treatments combining CA and Protein Kinase C agonists result in an even larger increase of abundance while conserving noise levels with a highly non-linear increase in variance of up to 63× untreated controls. Finally, drug combinations provide non-antagonistic combinatorial tuning of gene expression noise and map a noise phase space for future applications with viral and synthetic gene vectors. Active remodeling of nucleosomes and BAF-mediated control of gene expression noise expand a toolbox for the future design and engineering of stochasticity in living systems.

Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional assays.

Twenty amino acid substitutions in the thymidine kinase (TK) of clinical herpes simplex virus type 1 strains were assessed for conferring acyclovir (ACV) resistance. Site-directed mutagenesis, cell-free protein synthesis and protein expression in Escherichia coli were performed to obtain recombinant TK proteins, which were authenticated by Western blotting. A modified enzyme-linked immunosorbent assay (ELISA) was carried out to determine the phosphorylation activity of the mutants towards 5-bromo-2'-deoxyuridine (BrdU). The activity against ACV and deoxythymidine (dT) was analyzed by high performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) following incubation of recombinant TK with ACV and dT. Using ELISA, seven substitutions (G61E, A93V, M121K, R163G, P173del, V238F, G264V) showing negative activity could be classified likely as resistance-related, eleven (Q15K, R20C, R32H, E43A, E43D, R89H, A156V, P269S, G271V, S276N, I326V) with high activity as natural polymorphisms, and two (N244H and N376stop) with low phosphorylation activity. Since the N244H protein did not show any activity towards ACV, but activity towards dT using HPLC/UV, it was classified as TK with altered substrate specificity. In conclusion, the ELISA determining activity towards BrdU is suitable for the characterization of substitutions regarding their significance for resistance. Ambiguous results can be re-assessed by HPLC/UV, which classifies TK with altered substrate specificity.

Similarity in viral and host promoters couples viral reactivation with host cell migration.

Viral-host interactomes map the complex architecture of an evolved arms race during host cell invasion. mRNA and protein interactomes reveal elaborate targeting schemes, yet evidence is lacking for genetic coupling that results in the co-regulation of promoters. Here we compare viral and human promoter sequences and expression to test whether genetic coupling exists and investigate its phenotypic consequences. We show that viral-host co-evolution is imprinted within promoter gene sequences before transcript or protein interactions. Co-regulation of human immunodeficiency virus (HIV) and human C-X-C chemokine receptor-4 (CXCR4) facilitates migration of infected cells. Upon infection, HIV can actively replicate or remain dormant. Migrating infected cells reactivate from dormancy more than non-migrating cells and exhibit differential migration-reactivation responses to drugs. Cells producing virus pose a risk for reinitiating infection within niches inaccessible to drugs, and tuning viral control of migration and reactivation improves strategies to eliminate latent HIV. Viral-host genetic coupling establishes a mechanism for synchronizing transcription and guiding potential therapies.

Database on natural polymorphisms and resistance-related non-synonymous mutations in thymidine kinase and DNA polymerase genes of herpes simplex virus types 1 and 2.

The use of genotypic resistance testing of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is increasing because the rapid availability of results significantly improves the treatment of severe infections, especially in immunocompromised patients. However, an essential precondition is a broad knowledge of natural polymorphisms and resistance-associated mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes, of which the DNA polymerase (Pol) enzyme is targeted by the highly effective antiviral drugs in clinical use. Thus, this review presents a database of all non-synonymous mutations of TK and DNA pol genes of HSV-1 and HSV-2 whose association with resistance or natural gene polymorphism has been clarified by phenotypic and/or functional assays. In addition, the laboratory methods for verifying natural polymorphisms or resistance mutations are summarized. This database can help considerably to facilitate the interpretation of genotypic resistance findings in clinical HSV-1 and HSV-2 strains.

Resistance testing of clinical herpes simplex virus type 2 isolates collected over 4 decades.

There is only little information about the role of mutations of the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type 2 (HSV-2) for the development of antiviral resistance. In this study, the polymorphism of TK and DNA pol genes was examined in 82 clinical isolates collected routinely between 1973 and 2013. If novel, presently unclear or resistance-related mutations were found, the resistance phenotype against acyclovir (ACV) and foscarnet (FOS) was analyzed. The four novel amino acid changes G150D, A157T, R248W, L342W and the hitherto phenotypically unclear substitution T131M within the TK gene were identified as natural polymorphisms. Within the DNA pol gene, 17 novel substitutions and the to-date unclear change R628C were characterized as part of natural gene polymorphism. Two novel DNA pol mutations were linked to resistance (M910T) and weak susceptibility to ACV (684 insertion ED), respectively. In one isolate, the genomic cause of ACV resistance could not be identified. Phylogenetic analysis including sequences of this study and of the GenBank revealed a hierarchy of mutation clusters in TK displaying G39E as first common mutation step, followed by N78D and L140F. In conclusion, the present findings allow a deeper insight in the variability of HSV-2 TK and DNA pol genes. The most common substitution G39E can be excluded as unique cause of HSV-2 resistance. This study supports once more the importance of phenotypic adjustment of genotypic results to enhance the clinical utility of genotypic findings.

Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance.

A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance.

Drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate.

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.