Britt A. Glaunsinger’s research while affiliated with University of California, Berkeley and other places

What is this page?


This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Publications (150)


Enhancers and genome conformation provide complex transcriptional control of a herpesviral gene
  • Article

November 2024

·

8 Reads

Molecular Systems Biology

·

Leah Gulyas

·

Xiaowen Mao

·

[...]

·

Britt A Glaunsinger

Complex transcriptional control is a conserved feature of both eukaryotes and the viruses that infect them. Despite viral genomes being smaller and more gene dense than their hosts, we generally lack a sense of scope for the features governing the transcriptional output of individual viral genes. Even having a seemingly simple expression pattern does not imply that a gene’s underlying regulation is straightforward. Here, we illustrate this by combining high-density functional genomics, expression profiling, and viral-specific chromosome conformation capture to define with unprecedented detail the transcriptional regulation of a single gene from Kaposi’s sarcoma-associated herpesvirus (KSHV). We used as our model KSHV ORF68 – which has simple, early expression kinetics and is essential for viral genome packaging. We first identified seven cis-regulatory regions involved in ORF68 expression by densely tiling the ~154 kb KSHV genome with dCas9 fused to a transcriptional repressor domain (CRISPRi). A parallel Cas9 nuclease screen indicated that three of these regions act as promoters of genes that regulate ORF68. RNA expression profiling demonstrated that three more of these regions act by either repressing or enhancing other distal viral genes involved in ORF68 transcriptional regulation. Finally, we tracked how the 3D structure of the viral genome changes during its lifecycle, revealing that these enhancing regulatory elements are physically closer to their targets when active, and that disrupting some elements caused large-scale changes to the 3D genome. These data enable us to construct a complete model revealing that the mechanistic diversity of this essential regulatory circuit matches that of human genes.


The SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity
  • Preprint
  • File available

August 2024

·

30 Reads

Type I interferons (IFN-Is) are essential for antiviral immunity but must be tightly regulated. The conserved transcriptional repressor SP140 inhibits interferon beta ( Ifnb1 ) expression via an unknown mechanism. We find that SP140 does not repress Ifnb1 transcription but instead negatively regulates Ifnb1 mRNA stability by directly repressing the expression of a previously uncharacterized regulator we call RESIST (REgulated Stimulator of Interferon via Stabilization of Transcript, previously annotated as Annexin-2 Receptor). RESIST promotes Ifnb1 mRNA stability by counteracting Ifnb1 mRNA destabilization mediated by the Tristetraprolin (TTP) family of RNA-binding proteins and the CCR4-NOT deadenylase complex. SP140 localizes within nuclear bodies, punctate structures that play important roles in silencing DNA virus gene expression in the nucleus. Consistent with this observation, we found that SP140 inhibits replication of the gammaherpesvirus MHV68. The antiviral activity of SP140 was independent of its ability to regulate Ifnb1 . Our results establish dual antiviral and interferon regulatory functions for SP140 and identify the SP140-RESIST pathway as a novel regulator of Ifnb1 mRNA stability.

Download


The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

January 2024

·

13 Reads

Many stressors, including viral infection, induce a widespread suppression of cellular RNA polymerase II (RNAPII) transcription, yet the mechanisms underlying transcriptional repression are not well understood. Here we find that a crucial component of the RNA polymerase II holoenzyme, general transcription factor IIB (TFIIB), is targeted for post-translational turnover by two pathways, each of which contribute to its depletion during stress. Upon DNA damage, translational stress, apoptosis, or lytic infection with the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV), TFIIB is cleaved by activated caspase-3, leading to preferential downregulation of pro-survival genes. TFIIB is further targeted for rapid proteasome-mediated turnover by the E3 ubiquitin ligase TRIM28. KSHV counteracts proteasome-mediated turnover of TFIIB, thereby preserving a sufficient pool of TFIIB for transcription of viral genes. Thus, TFIIB may be a lynchpin for transcriptional outcomes during stress and a key target for nuclear replicating DNA viruses that rely on host transcriptional machinery. Graphical Abstract


Coding and non-coding elements comprise a regulatory network controlling transcription in Kaposi's sarcoma-associated herpesvirus

July 2023

·

41 Reads

Gene regulation in eukaryotes relies on many mechanisms for optimal expression, including both protein transcription factors and DNA regulatory elements. CRISPR-based screens of both protein coding genes and non-coding regions have allowed identification of these transcriptional networks in human cells. Double-stranded DNA viruses also invoke human-like regulation to control transcription of viral genes that are required at different stages of the viral lifecycle. Here, we applied CRISPR-based tools to dissect regulation of a viral gene at high resolution in the oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), whose compact, densely encoded genome provides unique challenges and opportunities for studying transcriptional networks. Through a combination of CRISPR-interference (CRISPRi) and Cas9 nuclease screening, we mapped a novel regulatory network comprised of coding and noncoding elements that influence expression of the essential KSHV protein ORF68 at early and late stages of the viral lifecycle. ORF68 encodes an essential protein involved in packaging the replicated viral DNA into nascent capsids. Although ORF68 expression initiates early in the viral lifecycle, we found that it is primarily required at later times. This work demonstrates the ability to exhaustively identify features controlling a given locus, essentially capturing a complete viral regulatory circuit that functions within the human nucleus to control transcription.


High-throughput discovery and characterization of viral transcriptional effectors in human cells

June 2023

·

33 Reads

·

16 Citations

Cell Systems

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across ∼1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in viral interferon regulatory factors (VIRFs), and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in Kaposi's sarcoma-associated herpesvirus (KSHV). For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.


The vTA complex is associated with numerous cellular and viral factors during infection
(A) TurboID was tethered to the C-terminus of ORF18. ORF18 directly contacts ORF30, ORF31, ORF66, and ORF34 and is proximal to ORF24 and RNA polymerase II. (B) Infectious virion production from reactivated iSLKs was measured by viral supernatant transfer onto HEK293T cells using flow cytometry for the GFP-expressing virus. Data are from three independent biological replicates and statistics are calculated using an unpaired t test. * = P<0.05. (C) Overlap between proteins identified by mass spectrometry with ≥2 unique peptides that demonstrated >2-fold increase in the ORF18-TurboID + biotin test condition compared to the three negative controls, across 2 biological replicates. Forty-five high confidence hits were specifically enriched in ORF18-TurboID + biotin sample using these filtering criteria. (D) STRING protein-protein interaction network of high-confidence cellular proteins (above) and KSHV proteins (below). ORF18 is shown in dark blue. Known interactors of ORF18 are in light blue, and all other proteins are shown in grey. Proteins with nuclear localization annotated in UniProt are outlined in gold. (E) Gene ontology enrichment analysis of cellular ORF18-TurboID hits. All major enriched functional classes are shown.
Functional analysis of ORF18-interacting proteins
(A) Schematic of the late gene reporter virus used to measure late gene expression by flow cytometry. The reporter construct was cloned into a region of the vector backbone of the KSHV BAC16. The K8.1 late gene promoter drives expression of eGFP, and mIFP is constitutively expressed from the EF-1a promoter. (B) Late gene reporter signal for CRISPR knockouts of viral genes at 72 h post reactivation. Knockouts were normalized against the non-targeting control (NT) within each replicate. Data are from three independent biological replicates, **** = P<0.0001. P values calculated from ordinary one-way ANOVA test. (C) Late gene reporter signal for CRISPR knockouts of cellular genes at 72 h post reactivation. Knockouts were normalized against the non-targeting control (NT) within each replicate. Data are from three independent biological replicates, **** = P<0.0001, * = P<0.05. P values calculated from ordinary one-way ANOVA test. (D) Early gene reporter signal for CRISPR knockouts of viral and cellular genes at 48 h post reactivation. Knockouts were normalized against the non-targeting control (NT) within each replicate. Data are from three independent biological replicates, **** = P<0.0001. P values calculated from ordinary one-way ANOVA test. (E) Viral DNA replication was measured using qPCR to compare reactivated to non-reactivated cells for each CRISPR knockout line. Data are from three independent biological replicates, * = P<0.05. P values calculated from ordinary one-way ANOVA test.
SiRNA-based depletion of select TurboID hits suggests a generalized role for PABC proteins in viral gene expression
(A) Representative western blots of siRNA-treated WT iSLK cell lysates at 48 h post reactivation showing the efficiency of XRN1, CASK, and PABPC1 depletion as well as levels of the representative late protein, K8.1, and early protein, ORF6. Vinculin serves as a loading control. (B) Representative western blots of WT iSLK cells at 48 h post reactivation that were depleted of PABPC1 and PABPC4 individually or in tandem by siRNA treatment. Levels of late proteins K8.1 and ORF26 are shown, as are levels of early proteins ORF6, ORF68, and ORF59. Vinculin serves as a loading control.
Loss of ORF29 impairs late gene transcription
(A) Western blot showing the expression kinetics of ORF29 relative to the early proteins ORF59, ORF6, and ORF68, and the late protein K8.1 in reactivated WT iSLK cells. (B) Infectious virion production from reactivated iSLK cells was measured by viral supernatant transfer using flow cytometry. Data are from three independent biological replicates, **** = P<0.0001. P values calculated from ordinary one-way ANOVA test. (C) Representative western blots of lysates harvested from iSLK cells at 72 h post reactivation. Levels of the late proteins, K8.1 and ORF26, are shown, as are levels of the early proteins, ORF6, ORF59, and ORF68. GAPDH serves as a loading control. (D) RNA levels of the indicated genes were measured by RTqPCR. Reactivated cells were harvested at 48 h post reactivation. Data are from three independent biological replicates, normalized to WT for each transcript, **** = P<0.0001, ** = P<0.005, * = P<0.05. P values calculated from ordinary one-way ANOVA test. (E) RNA levels of the indicated genes were measured by RTqPCR. Reactivated cells were harvested at 72 h post reactivation. Data are from three independent biological replicates, normalized to WT for each transcript, **** = P<0.0001, ** = P<0.005, * = P<0.05. P values calculated from ordinary one-way ANOVA test. (F) RNA levels of the indicated genes were measured by RTqPCR. Reactivated cells were harvested at 24 h post reactivation. Data are from three independent biological replicates, normalized to WT for each transcript, **** = P<0.0001, ** = P<0.005, * = P<0.05. P values calculated from ordinary one-way ANOVA test. (G) Viral DNA replication was measured using qPCR in cells harvested 24, 48, and 72 h post reactivation. Fold DNA replication values were calculated by comparing reactivated to non-reactivated cells. Data are from three independent biological replicates, * = P<0.05. P values calculated from ordinary one-way ANOVA test.
Complementation with ORF29 functional mutants fails to rescue late gene transcription in the background of an ORF29.stop virus
(A) Schematic of the ORF29 coding sequence. Positions of the Walker A and Walker B sequences are indicated, as are the residues that have previously been implicated in magnesium coordination and nuclease activity. The point mutants used in subsequent experiments are indicated in red. (B) Infectious virion production from reactivated iSLK cells was measured by viral supernatant transfer using flow cytometry. Data are from three independent biological replicates, **** = P<0.0001. P values calculated from ordinary one-way ANOVA test. (C) Representative western blots of lysates harvested from iSLK cells harboring the ORF29.stop virus complemented with ORF29 variants at 72 h post reactivation. Levels of the late protein K8.1 and early proteins ORF59 and ORF6 are shown. GAPDH serves as a loading control. (D) RNA levels of the indicated genes were measured by RTqPCR. Reactivated cells were harvested at 72 h post reactivation. Data are from three independent biological replicates, normalized to WT for each transcript, ** = P<0.005. P values calculated from ordinary one-way ANOVA test. (E) Viral DNA replication was measured using qPCR in cells harvested 72 h post reactivation. Fold DNA replication values were calculated by comparing reactivated to non-reactivated cells. Data are from three independent biological replicates. P values calculated from ordinary one-way ANOVA test.

+1

The viral packaging motor potentiates Kaposi’s sarcoma-associated herpesvirus gene expression late in infection

April 2023

·

51 Reads

·

4 Citations

β- and γ-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II and an unknown set of additional factors. We employed proximity labeling coupled with mass spectrometry, followed by CRISPR and siRNA screening to identify proteins functionally associated with the Kaposi’s sarcoma-associated herpesvirus (KSHV) late gene transcriptional complex. These data revealed that the catalytic subunit of the viral DNA packaging motor, ORF29, is both dynamically associated with the viral transcriptional activator complex and potentiates gene expression late in infection. Through genetic mutation and deletion of ORF29, we establish that its catalytic activity potentiates viral transcription and is required for robust accumulation of essential late proteins during infection. Thus, we propose an expanded role for ORF29 that encompasses its established function in viral packaging and its newly discovered contributions to viral transcription and late gene expression in KSHV.


Murine Gammaherpesvirus 68 ORF45 Stimulates B2 Retrotransposon and Pre-tRNA Activation in a Manner Dependent on Mitogen-Activated Protein Kinase (MAPK) Signaling

February 2023

·

44 Reads

·

2 Citations

Microbiology Spectrum

RNA polymerase III (RNAPIII) transcribes a variety of noncoding RNAs, including tRNA (tRNA) and the B2 family of short interspersed nuclear elements (SINEs). B2 SINEs are noncoding retrotransposons that possess tRNA-like promoters and are normally silenced in healthy somatic tissue. Infection with the murine gammaherpesvirus MHV68 induces transcription of both SINEs and tRNAs, in part through the activity of the viral protein kinase ORF36. Here, we identify the conserved MHV68 tegument protein ORF45 as an additional activator of these RNAPIII loci. MHV68 ORF45 and ORF36 form a complex, resulting in an additive induction RNAPIII and increased ORF45 expression. ORF45-induced RNAPIII transcription is dependent on its activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway, which in turn increases the abundance of the RNAPIII transcription factor Brf1. Other viral and nonviral activators of MAPK/ERK signaling also increase the levels of Brf1 protein, B2 SINE RNA, and tRNA, suggesting that this is a common strategy to increase RNAPIII activity. IMPORTANCE Gammaherpesviral infection alters the gene expression landscape of a host cell, including through the induction of noncoding RNAs transcribed by RNA polymerase III (RNAPIII). Among these are a class of repetitive genes known as retrotransposons, which are normally silenced elements and can copy and spread throughout the genome, and transfer RNAs (tRNAs), which are fundamental components of protein translation machinery. How these loci are activated during infection is not well understood. Here, we identify ORF45 from the model murine gammaherpesvirus MHV68 as a novel activator of RNAPIII transcription. To do so, it engages the MAPK/ERK signaling pathway, which is a central regulator of cellular response to environmental stimuli. Activation of this pathway leads to the upregulation of a key factor required for RNAPIII activity, Brf1. These findings expand our understanding of the regulation and dysregulation of RNAPIII transcription and highlight how viral cooption of key signaling pathways can impact host gene expression.


The viral packaging motor potentiates late gene expression in Kaposi's sarcoma-associated herpesvirus

January 2023

·

13 Reads

β- and γ-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II and an unknown set of additional factors. We employed proximity labeling coupled with mass spectrometry, followed by CRISPR and siRNA screening to identify proteins functionally associated with the Kaposi’s sarcoma-associated herpesvirus (KSHV) late gene transcriptional complex. These data revealed that the catalytic subunit of the viral DNA packaging motor, ORF29, is both dynamically associated with the viral transcriptional activator complex and potentiates late gene expression. Through genetic mutation and deletion of ORF29, we establish that its catalytic activity potentiates viral transcription and is required for robust accumulation of essential late proteins during infection. Thus, we propose an expanded role for ORF29 that encompasses its established function in viral packaging and its newly discovered contributions to viral transcription and late gene expression in KSHV. Author summary β- and γ-herpesviruses express a class of genes essential for completion of the viral life cycle late during infection. A specialized complex of viral transcriptional activator proteins drives expression of these late genes in a manner dependent on viral genome replication, although the mechanisms and regulation of this process are largely unknown. Here, we identified factors that physically and functionally associate with the late gene transcription complex and unexpectedly found that the viral DNA packaging motor in Kaposi’s sarcoma-associated herpesvirus (KSHV) contributes to late gene expression. We show that the catalytic activity of this protein is not only required for genomic packaging but also for the robust expression of late genes to ensure the successful production of progeny virions. Thus, late gene transcription is mechanistically linked to the conserved processes of viral genome replication and packaging.


Figure 2. In vitro characterization of SOX DNA processing mutants. (A) A total of 8 SOX mutants were grouped into three categories: catalytic residues, putative DNA binding residues, and other highly conserved residues. (B) Crystal structure of the KSHV SOX (PDB 3POV). Highlighted box shows catalytic residues represented in pink, conserved residues in green, and putative DNA binding residues in orange. (C) SOX mutant K d values were grouped according to their putative functions and plotted. K d and error bars were derived from fitting graphs to single binding models for three independent measurements. (D) Catalytic efficiencies were determined for all eight SOX mutants (25 nM) in the presence of a 25 bp ssDNA probe (1 nM) derived from the viral LANA gene containing a 5' phosphate and a 3' fluorophore. Experiments were performed in triplicate and in substrate limiting conditions. (E) The exonuclease activity of WT and mutated versions of SOX were measured using a continuous fluorescence assay. Excess amount of enzyme was used (250 nM) with a limiting amount of substrate (0.01 nM) and plotted using percent digestion as a function of time.
Figure 6. SOX end processing mutants produce fewer infectious virions. Cartoon representation of SOX end processing mutants resulting in the production of fewer infectious virions.
DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production

December 2022

·

39 Reads

·

4 Citations

Nucleic Acids Research

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.


Citations (45)


... KRAB domains are the best repressors across context, with the KRAB domain from ZNF705F being a notable find, as it has higher efficacy than the more commonly used KRAB from ZNF10. Interestingly, a strong transcriptionally activating KRAB domain was also found, highlighting how informative such large unbiased screenings can be [30][31][32]. ...

Reference:

Bridging bioengineering and epigenetics: from technical innovations to clinical applications
High-throughput discovery and characterization of viral transcriptional effectors in human cells
  • Citing Article
  • June 2023

Cell Systems

... 44) ORF29 is involved in infectious virus production, viral genome replication, and expression of viral late genes. 45) ORF7 interacts with ORF29 and ORF67.5, however ORF29 and ORF67.5 do not interact directly. 46) ORF7 can translocate to the nucleus, whereas ORF29 alone and ORF67.5 alone lacks this ability. ...

The viral packaging motor potentiates Kaposi’s sarcoma-associated herpesvirus gene expression late in infection

... Using long-read RNA-seq, she identified hundreds of host isoformswitching events dependent on virus-activated B2 SINEs and determined that these switching events were enriched for transcripts that encode innate immune factors. This work points to a mechanism that drives host-isoform switching during viral infection through the co-option of B2 SINE elements [21]. ...

Murine Gammaherpesvirus 68 ORF45 Stimulates B2 Retrotransposon and Pre-tRNA Activation in a Manner Dependent on Mitogen-Activated Protein Kinase (MAPK) Signaling

Microbiology Spectrum

... Although BGLF5 is not known to play a direct role in late gene transcription, it is essential for processing of viral DNA for encapsidation, and dysregulation of viral DNA processing may impact the efficiency of late gene transcription from newly replicated viral DNA. 33,[36][37][38][39] We next compared host transcript levels in early lytic and late lytic DFLRΔBGLF5 LCLs to those seen in WT DFLR LCLs. Remarkably, extensive host shutoff was observed even in the absence of BGLF5 ( Figure 7A and Table S3). ...

DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production

Nucleic Acids Research

... These include not only cytoskeletal changes, but also alterations in the 3D structure of the genome and the occurrence of cell and DNA damage (13,14). This implies that many differentially regulated transcripts in virus-infected cells may not have discernible functions and may be the result of cytopathic effects or perturbation of RNA polymerase II function that is affected by various viruses (15). ...

RNA polymerase II subunit modulation during viral infection and cellular stress
  • Citing Article
  • October 2022

Current Opinion in Virology

... The binding pocket of Telomere-binding protein OPG077 was detected by using the CB-DOCK2 server using ATP as a ligand. The CB-DOCK2 server predicted 05 cavities (Supplementary table S18 has been the subject of research to better understand poxvirus biology and as a target for antiviral strategies [116][117][118] . ...

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

... During alphaherpesvirus infection, many host gene-expressions were controlled depending on the viral lifecycle. For example, HSV-1 infection could regulate host gene-expression through polymerase II and III [4,5]. In the case of host genes egr-1 and c-fos, which is expressed instantaneously, depends on neuronal activity and the protein expression level is altered by HSV-1 infection [6,7]. ...

Manipulation of RNA polymerase III by Herpes Simplex Virus-1

... The H213L mutation in the putative DNA binding domain of EBV vUNG disrupts interaction with EBV vPOL in the context of reactivation, and this mutant exhibited a reactivation defect (24). A recent study with KSHV reported that reactivation of KSHV BAC DNA containing mutations in the putative DNA binding domain (H210L) of vUNG (ORF46), but not the putative enzymatic domain (Q87L/D88N), resulted in a defect in viral DNA synthesis and virus production (36). A caveat to these previous studies is that the loss of biochemical functions was not validated. ...

A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains

... (LAV) candidates, OTS-206 and OTS-228, employing the elegant OTS method [3], which involves introducing synonymous codon changes to increase the likelihood of premature termination codons, thereby reducing viral fitness. They also introduced additional modifications such as disabling Nsp1 translational repression [4], removing open reading frames (ORFs) 6, 7ab, and 8 [5][6][7][8], and optimizing the spike polybasic cleavage site [9] to further enhance safety and immunogenicity. Preclinical studies in animal models (mouse and golden Syrian hamster) revealed that OTS-228 demonstrated an optimal safety profile, exhibiting no detectable side effects or transmission in preclinical animal models. ...

The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

Cell Reports