hsv 1 strain hf  (ATCC)

Journal: bioRxiv

Article Title: In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation

doi: 10.64898/2026.01.06.697999

Figure Lengend Snippet: 17ΔA gene expression following the application of reactivation stimulus wortmannin. A. wt and 17ΔA HSV-1 genome copies in quiescently infected LUHMES cells (MOI of 0.1). Relative genome load was determined by qPCR copy number of DNA polymerase normalized to that of host GAPDH. (n=3). Statistical significance between wt and 17ΔA was calculated by unpaired two-tailed Student’s t-tests with equal variance. B. VP16 gene expression in 17ΔA was quantified by qRT-PCR in LUHMES cells. Cells were quiescently infected at an MOI of 0.2 and total RNA was extracted at 8 dpi (latency), 1, 3, 6 and 24 hpr. Gene expression was quantified using primers and probes listed in Table S2. Relative values for each gene were normalized to host GAPDH expression and were plotted as fold change in expression relative to latency (set to 1). n=3. * p <0.05, ** p <0.005, *** p <0.0005 following Student’s t-tests. C . Expression of representative genes from each kinetic class were quantified by RT-qPCR as described in panel B. D . Comparison between gene expression of wt and 17ΔA at 1 and 6 hpr. E. Luciferase reporter constructs were generated to test the activation of VP16 promoter in neurons using the promoterless pGL3 basic vector, pGL3 basic-LTE (n.t.118,889-119,478), VP16 promoter only, VP5 promoter only, and VP16 or VP5 promoter inserted into the LTE plasmid. F. Luciferase reporter assays were performed in mice N2a cells. All transfections were completed in triplicate wells and were repeated three times biologically. All luciferase values were normalized to the pGL3 basic vector (set to 1). Statistics were determined by unpaired two-tailed Student’s t-tests with unequal variance.

Article Snippet: Viral genome copy numbers were determined by qPCR using primers and probe specific for HSV-1 DNA polymerase (Table S2). qPCRs were performed using TaqManTM Fast Universal PCR Master Mix (2×), no AmpEraseTM UNG (Applied biosystems 4352042) on an Agilent AriaMx Real-Time PCR machine.

Techniques: Gene Expression, Infection, Two Tailed Test, Quantitative RT-PCR, Expressing, Comparison, Luciferase, Construct, Generated, Activation Assay, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation

doi: 10.64898/2026.01.06.697999

Figure Lengend Snippet: Luciferase reporter constructs were generated to test the enhancer-blocking ability of the predicted CTCF insulator sites. A . Schematic representation of CTCFBSDB predicted CTCF binding sites in the UL region of the HSV-1 genome near VP16. B . Representative orientations for each of the constructs tested include the commercially available pGL3-control vector with an SV40 promoter and a luciferase gene as the plasmid backbone; the LTE inserted into the control vector; each putative CTCF-binding site identified in Table S1 together with ∼150-350 bp flanking sequences inserted downstream of the LTE. C . Luciferase reporter assays were performed in N2a as a representative neuronal cell line. All transfections were completed in triplicate. Data represent at least three biological replicates. All luciferase values were normalized to the pGL3-control vector as a fold change in expression relative to control (set to 1). CTRL2 served as a positive control with known LTE-blocking activity in N2a. Statistical comparisons were done on fold changes between the LTE construct and the LTE-CTCF site. * p <0.05, ** p <0.005, *** p <0.0005 by unpaired two-tailed Student s t-tests with unequal variance.

Article Snippet: Viral genome copy numbers were determined by qPCR using primers and probe specific for HSV-1 DNA polymerase (Table S2). qPCRs were performed using TaqManTM Fast Universal PCR Master Mix (2×), no AmpEraseTM UNG (Applied biosystems 4352042) on an Agilent AriaMx Real-Time PCR machine.

Techniques: Luciferase, Construct, Generated, Blocking Assay, Binding Assay, Control, Plasmid Preparation, Transfection, Expressing, Positive Control, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation

doi: 10.64898/2026.01.06.697999

Figure Lengend Snippet: CTCF and cohesin proteins are enriched on CTUL1 during HSV-1 latency but evicted by 2 hpr. Protein binding was determined by ChIP or CUT&RUN and quantified by qPCR using primers and probes specific to the given gene regions of HSV-1 (Table S2). All relative values for CTCF enrichment were normalized to those of the non-specific binding control IgG and were plotted as fold enrichment relative to IgG (set to 1). n=3-5. A. LUHMES cells were infected at MOI of 0.3 and harvested at 8 dpi for ChIP. CTCF binding in LUHMES was validated at the host positive control region H19/Igf2. qPCR primers specific to CTUL1 or CTRL2 were used to quantitate CTCF enrichment (Table S2). gC served as a negative control of CTCF binding on HSV-1 genome. Each ChIP assay represented one 6-well plate of cells. n=3. Binding significance was determined by Student’s t-tests. * p <0.05. B. CUT&RUN-qPCR was performed with quiescently infected LUHMES cells as described above. CTCF binding significance was determined by one-way analysis of variance (ANOVA). n=3. C. ChIP-qPCR was done with the anti-STAG2 antibody on latently infected LUHMES. STAG2 binding in LUHMES was validated at a host positive control region on human Chromosome 1, as previously reported (Table S2). Primers specific for CTUL1 were used to quantitate STAG2 on each site following ChIP. n=4. gC served as a negative control on the viral genome without STAG2 enrichment. Binding significance was determined by unpaired two-tailed Student’s t-tests. * p <0.05, ** p <0.005. D . LUHMES cells were infected at MOI of 0.3 for 8 days and subjected to wortmannin treatment for 2 hours, followed by ChIP-qPCR. CTCF bound/input values at CTUL1 and CTRL2 were normalized to bound/input at host H19/Igf2. Then, fold change was calculated between 2 hpr and latency (set to 1). * p <0.05, ** p <0.005 by Student’s t-test. E . ChIP-qPCR done with anti-STAG2 antibody on LUHMES subjected to wortmannin treatment for 2 hours. Fold change values plotted were calculated by setting latency to 1. n=3. F . Luciferase reporter constructs were generated to test CTUL1 as an enhancer-blocker to the VP16 promoter in the presence of the CTUL1 insulator binding site and the LTE. A 294-bp random sequence was inserted in place of CTUL1. G. Transient transfections were performed in N2a cells. All transfections were completed in triplicate wells and were repeated three times biologically. All luciferase values were normalized to the pGL3-basic vector (set to 1). * p< 0.05 by unpaired two-tailed Student’s t-tests with unequal variance.

Article Snippet: Viral genome copy numbers were determined by qPCR using primers and probe specific for HSV-1 DNA polymerase (Table S2). qPCRs were performed using TaqManTM Fast Universal PCR Master Mix (2×), no AmpEraseTM UNG (Applied biosystems 4352042) on an Agilent AriaMx Real-Time PCR machine.

Techniques: Protein Binding, Binding Assay, Control, Infection, Positive Control, Negative Control, ChIP-qPCR, Two Tailed Test, Luciferase, Construct, Generated, Sequencing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation

doi: 10.64898/2026.01.06.697999

Figure Lengend Snippet: A model illustrating how CTCF insulators regulate VP16 de novo gene expression upon HSV-1 reactivation. Beige line indicates HSV-1 genome. Rectangles on the genome denote elements regulatory elements: blue: LAT enhancer; red/green: VP16 promoter; yellow: insulators. Red line denotes transcriptional repression while green line denotes activation. In latency, CTUL1 and another unidentified insulator are enriched with CTCF protein and anchored by the cohesin complex. This structure insulates LTE from turning on the VP16 promoter. In reactivation, CTCF proteins evict from the insulators and cohesin moves away. The loss of enhancer-blocking activity allows LTE to turn on the VP16 promoter.

Article Snippet: Viral genome copy numbers were determined by qPCR using primers and probe specific for HSV-1 DNA polymerase (Table S2). qPCRs were performed using TaqManTM Fast Universal PCR Master Mix (2×), no AmpEraseTM UNG (Applied biosystems 4352042) on an Agilent AriaMx Real-Time PCR machine.

Techniques: Gene Expression, Activation Assay, Blocking Assay, Activity Assay