A Generally Applicable CRISPR/Cas9 Screening Technique to Identify Host Genes Required for Virus Infection as Applied to Human Cytomegalovirus (HCMV) Infection of Epithelial Cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens enable virus-host genetic screens to be undertaken in a more robust manner than previously possible and has had a tremendous impact in the field of virus study. Researchers can take advantage of the power of CRISPR genetic screens to discover virus-host interaction genes including host receptors and signaling molecules (Bazzone et al., mBio 10 (1): e02734-18, 2019; E et al., Proc Natl Acad Sci U S A 116(14):7043-7052, 2019; McDougall et al., Curr Opin Virol 29:87-100, 2018; Savidis et al., Cell Rep 16(1):232-246, 2016). In principle, lysis of cells late in the virus infection cycle allows one to screen for essential genes using pooled single-guide RNAs (sgRNAs) that collective target an entire host cell genome simply by identifying mutant cells that are resistant to virus-induced cell death. Here we focus on using this technique on epithelial cells to identify host targets required for human cytomegalovirus (HCMV) infection.

Toll-like receptor-mediated innate immunity against herpesviridae infection: a current perspective on viral infection signaling pathways.

BACKGROUND: In the past decades, researchers have demonstrated the critical role of Toll-like receptors (TLRs) in the innate immune system. They recognize viral components and trigger immune signal cascades to subsequently promote the activation of the immune system. MAIN BODY: Herpesviridae family members trigger TLRs to elicit cytokines in the process of infection to activate antiviral innate immune responses in host cells. This review aims to clarify the role of TLRs in the innate immunity defense against herpesviridae, and systematically describes the processes of TLR actions and herpesviridae recognition as well as the signal transduction pathways involved. CONCLUSIONS: Future studies of the interactions between TLRs and herpesviridae infections, especially the subsequent signaling pathways, will not only contribute to the planning of effective antiviral therapies but also provide new molecular targets for the development of antiviral drugs.

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism.

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

A Comparative Study of Single and Dual Perfusion During End-ischemic Subnormothermic Liver Machine Preservation.

BACKGROUND: It remains controversial if arterial perfusion in addition to portal vein perfusion during machine preservation improves liver graft quality. Comparative studies using both techniques are lacking. We studied the impact of using single or dual machine perfusion of donation after circulatory death rat livers. In addition, we analyzed the effect of pulsatile versus continuous arterial flow. METHODS: Donation after circulatory death rat livers (n = 18) were preserved by 6 hours cold storage, followed by 1 hour subnormothermic machine perfusion (20°C, pressure of 40/5 mm Hg) and 2 hours ex vivo warm reperfusion (37°C, pressure of 80/11 mm Hg, 9% whole blood). Machine preservation was either through single portal vein perfusion (SP), dual pulsatile (DPP), or dual continuous perfusion (DCP) of the portal vein and hepatic artery. Hydrodynamics, liver function tests, histopathology, and expression of endothelial specific genes were assessed during 2 hours warm reperfusion. RESULTS: At the end of reperfusion, arterial flow in DPP livers tended to be higher compared to DCP and SP grafts. However, this difference was not significant nor was better flow associated with better outcome. No differences in bile production or alanine aminotransferase levels were observed. SP livers had significantly lower lactate compared to DCP, but not DPP livers. Levels of Caspase-3 and tumor necrosis factor-α were similar between the groups. Expression of endothelial genes Krüppel-like-factor 2 and endothelial nitric oxide synthase tended to be higher in dual perfused livers, but no histological evidence of better preservation of the biliary endothelium or vasculature of the hepatic artery was observed. CONCLUSIONS: This study shows comparable outcomes after using a dual or single perfusion approach during end-ischemic subnormothermic liver machine preservation.

A novel DDB2-ATM feedback loop regulates human cytomegalovirus replication.

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.

An E2F1-mediated DNA damage response contributes to the replication of human cytomegalovirus.

DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses.

Autophagy evasion in herpesviral latency.

Autophagy constitutes a major catabolic process for the quality control of internal proteins and organelles of eukaryotic cells, and is emerging as an essential part of the host antiviral defense. Many studies have shed light on the importance of autophagy in homeostasis, but it is not well understood how viruses co-opt the cellular autophagic pathway to establish virulence in vivo. Our recent study presents direct in vivo evidence for the key role of the anti-autophagic aspect of the virally encoded Bcl-2 proteins in the chronic infection of oncogenic gamma-herpesviruses and proposes that cellular autophagy may have a substantial effect on viral persistence and may influence the in vivo fitness of viruses. This discovery expands upon known antiviral activities of the autophagy machinery and also suggests new approaches for treating some virally induced diseases.

Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68.

Gamma-herpesviruses (gammaHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine gamma-herpesvirus 68 (gammaHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant gammaHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which gammaHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of gamma-herpesviruses.

Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68.

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.

Downregulation of autophagy by herpesvirus Bcl-2 homologs.

The critical role of the cellular autophagy pathway in viral infection and pathogenesis has become increasingly apparent. Mounting evidences suggest that viruses have developed different strategies to meticulously modulate intracellular autophagy for their own benefits, thereby either promoting efficient viral replication or facilitating viral persistence. While our understanding of these strategies is still in its incipient stage, recent advances demonstrate that gamma herpesvirus Bcl-2 homolog (vBcl-2), which protects virus-infected cells from apoptosis, also suppresses cellular autophagy pathway through its direct interaction with the autophagy protein Beclin1. Interestingly, vBcl-2 has evolved to harbor the enhanced anti-autophagic activity compared to its host counterpart, suggesting an important role of cellular autophagy in response to viral infection and virus-associated pathogenesis. Thus, a detailed study of vBcl-2-mediated regulation of autophagy signal transduction pathway may lead to a better understanding of not only how virus escapes from host innate immunity but also how autophagy regulates viral infection and environmental stresses.

Parathyroid hormone inhibits renal phosphate transport by phosphorylation of serine 77 of sodium-hydrogen exchanger regulatory factor-1.

Parathyroid hormone (PTH), via activation of PKC and/or protein kinase A, inhibits renal proximal tubular phosphate reabsorption by facilitating the internalization of the major sodium-dependent phosphate transporter, Npt2a. Herein, we explore the hypothesis that the effect of PTH is mediated by phosphorylation of serine 77 (S77) of the first PDZ domain of the Npt2a-binding protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1). Using recombinant polypeptides representing PDZ I, S77 of NHERF-1 is phosphorylated by PKC but not PKA. When expressed in primate kidney epithelial cells (BSC-1 cells), however, activation of either protein kinase phosphorylates S77, suggesting that the phosphorylation of PDZ I by PKC and PKA proceeds by different biochemical pathways. PTH and other activators of PKC and PKA dissociate NHERF-1/Npt2a complexes, as assayed using quantitative coimmunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation in mice. Murine NHERF-1-/- renal proximal tubule cells infected with adenovirus-GFP-NHERF-1 containing an S77A mutation showed significantly increased phosphate transport compared with a phosphomimetic S77D mutation and were resistant to the inhibitory effect of PTH compared with cells infected with wild-type NHERF-1. These results indicate that PTH-mediated inhibition of renal phosphate transport involves phosphorylation of S77 of the NHERF-1 PDZ I domain and the dissociation of NHERF-1/Npt2a complexes.

Sodium-hydrogen exchanger regulatory factor-1 interacts with mouse urate transporter 1 to regulate renal proximal tubule uric acid transport.

Sodium-hydrogen exchanger regulatory factor-1-deficient (NHERF-1(-/-)) mice demonstrate increases in the urinary excretion of phosphate, calcium, and uric acid associated with interstitial deposition of calcium in the papilla of the kidney. These studies examine the role of NHERF-1 in the tubular reabsorption of uric acid and regulation of mouse urate transporter 1 (mURAT1), a newly described transporter that is responsible for the renal tubular reabsorption of uric acid. In primary cultures of mouse renal proximal tubule cells, uric acid uptake was significantly lower in NHERF-1(-/-) cells compared with wild-type cells over a large range of uric acid concentrations in the media. Western immunoblotting revealed a 56 +/- 6% decrease in the brush border membrane (BBM) expression of mURAT1 in NHERF-1(-/-) compared with wild-type control kidneys (P < 0.05). Confocal microscopy confirmed the reduced apical membrane expression of mURAT1 in NHERF-1(-/-) kidneys and demonstrated mislocalization of mURAT1 to intracellular vesicular structures. Para-aminohippurate significantly inhibited uric acid uptake in wild-type cells (41 +/- 2%) compared with NHERF-1(-/-) cells (8.2 +/- 3%). Infection of NHERF-1(-/-) cells with adenovirus-green fluorescence protein-NHERF-1 resulted in significantly higher rates of uric acid transport (15.4 +/- 1.1 pmol/microg protein per 30 min) compared with null cells that were infected with control adenovirus-green fluorescence protein (7.9 +/- 0.3) and restoration of the inhibitory effect of para-aminohippurate (% inhibition 34 +/- 4%). These findings indicate that NHERF-1 exerts a significant effect on the renal tubular reabsorption of uric acid in the mouse by modulating the BBM abundance of mURAT1 and possibly other BBM uric acid transporters.

Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone.

Sodium-dependent phosphate transport in NHERF-1(-/-) proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1(-/-) cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.

Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1-/- renal proximal tubule cells and wild-type cells adapted to low-phosphate media.

The present experiments using primary cultures from renal proximal tubule cells examine two aspects of the regulation of sodium-dependent phosphate transport and membrane sodium-dependent phosphate transporter (Npt2a) expression by parathyroid hormone (PTH). Sodium-dependent phosphate transport in proximal tubule cells from wild-type mice grown in normal-phosphate media averaged 4.4 +/- 0.5 nmol.mg protein(-1).10 min(-1) and was inhibited by 30.5 +/- 8.6% by PTH (10(-7) M). This was associated with a 32.7 +/- 5.2% decrease in Npt2a expression in the plasma membrane. Proximal tubule cells from Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1)(-/-) mice had a lower rate of phosphate transport compared with wild-type cells and a significantly reduced inhibitory response to PTH. Wild-type cells incubated in low-phosphate media for 24 h had a higher rate of phosphate transport compared with wild-type cells grown in normal-phosphate media but a significantly blunted inhibitory response to PTH. These data indicate a role for NHERF-1 in mediating the membrane retrieval of Npt2a and the subsequent inhibition of phosphate transport in renal proximal tubules. These studies also suggest that there is a blunted phosphaturic effect of PTH in cells adapted to low-phosphate media.

Defective parathyroid hormone regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1-/- renal proximal tubule cells.

The present experiments using primary cultures of renal proximal tubule cells derived from wild-type and NHERF-1 knockout animals examines the regulation of NHE3 by phenylthiohydantoin (PTH) and the regulation of phosphate transport in response to alterations in the media content of phosphate. Forskolin (34.8 +/- 6.2%) and PTH (29.7 +/- 1.8%) inhibited NHE3 activity in wild-type proximal tubule cells but neither forskolin (-3.2 +/- 3.3%) nor PTH (-16.6 +/- 8.1%) inhibited NHE3 activity in NHERF-1(-/-) cells. Using adenovirus-mediated gene transfer, expression of NHERF-1 in NHERF-1(-/-) proximal tubule cells restored the inhibitory response to forskolin (28.2 +/- 3.0%) and PTH (33.2 +/- 3.9%). Compared with high phosphate media, incubation of wild-type cells in low phosphate media resulted in a 36.0 +/- 6.3% higher rate of sodium-dependent phosphate transport and a significant increase in the abundance of Npt2a and PDZK1. NHERF-1(-/-) cells, on the other hand, had lower rates of sodium-dependent phosphate uptake and low phosphate media did not stimulate phosphate transport. Npt2a expression was not affected by the phosphate content of the media in NHERF-1 null cells although low phosphate media up-regulated PDZK1 abundance. Primary cultures of mice proximal tubule cells retain selected regulatory pathways observed in intact kidneys. NHERF-1(-/-) proximal tubule cells demonstrate defective regulation of NHE3 by PTH and indicate that reintroduction of NHERF-1 repairs this defect. NHERF-1(-/-) cells also do not adapt to alterations in the phosphate content of the media indicating that the defect resides within the cells of the proximal tubule and is not dependent on systemic factors.