Human cytomegalovirus UL27 is not required for viral replication in human tissue implanted in SCID mice

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo

Role of HCMV genes in benzimidazole ribonucleoside antiviral drug action.

Both benzimidazole D- and L-ribonucleosides are potent inhibitors of human cytomegalovirus (HCMV) replication. The D-nucleosides TCRB and BDCRB [2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole and its 2-bromo analog] were previously shown to inhibit HCMV DNA cleavage and processing. Mutations identified in genes UL56 and UL89 which encode a putative HCMV terminase were responsible for this resistance. Two strains with mutations either in UL89 or in both UL89 and UL56, also had a mutation (L21F) in gene UL104. This open reading frame encodes a putative HCMV portal protein that interacts with HCMV terminase. To determine if UL104 was involved in drug action, a recombinant virus with the L21F mutation only was constructed using the genome of HCMV cloned as a bacterial artificial chromosome. This recombinant virus was sensitive to TCRB/BDCRB which established that this mutation alone was not sufficient for resistance and was not involved in drug action unless it compensates for conformational changes in the mutant UL89 and UL56 proteins. The L-nucleoside maribavir [2-isopropylamino-5,6-dichloro-1-(beta- L-ribofuranosyl) benzimidazole) acts by a different mechanism inhibiting DNA synthesis and nuclear egress of HCMV. To investigate this drug's action, a strain of resistant HCMV was isolated. A series of recombinant resistant-wild type viruses were constructed and tested for sensitivity in order to narrow the resistance locus in this large DNA virus. The resistance mutation (L335P) was ultimately mapped to HCMV open reading frame UL27. Since the function of this gene in HCMV replication cycle is unknown, its protein was studied and found to be a nuclear protein with its own nuclear localization signal. Two UL27 deletion viruses also were constructed and their phenotype studied. Results showed that UL27 is not essential for HCMV replication in cell culture because HCMV with UL27 deleted grew at the same rate as the wild-type virus. However, it consistently achieved one-half log10 lower titers. Viral DNA synthesis of the UL27 deletion viruses also was about two fold lower than the wild-type virus. Both UL27 deletion viruses were sensitive to ganciclovir and foscarnet but were resistant to maribavir, confirming the role of this open reading frame in maribavir mechanism of action.Ph.D.Health and Environmental SciencesPharmacologyPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123883/2/3106100.pd

Role of a Mutation in Human Cytomegalovirus Gene UL104 in Resistance to Benzimidazole Ribonucleosides

The benzimidazole d-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV “terminase” and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA

Lipoprotein N-Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

Although it has long been known that S. aureus forms triacylated Lpps, a lack of homologs to known N-acylation genes found in Gram-negative bacteria has until now precluded identification of the genes responsible for this Lpp modification. Here, we demonstrate N-terminal Lpp acylation and chemotype conversion to the tri-acylated state is directed by a unique acyl transferase system encoded by two noncontiguous staphylococci genes (lnsAB). Since triacylated Lpps stimulate TLR2 more weakly than their diacylated counterparts, Lpp N-acylation is an important TLR2 immunoevasion factor for determining tolerance or nontolerance in niches such as in the skin microbiota. The discovery of the LnsAB system expands the known diversity of Lpp biosynthesis pathways and acyl transfer biochemistry in bacteria, advances our understanding of Lpp structural heterogeneity, and helps differentiate commensal and noncommensal microbiota.Bacterial lipoproteins (Lpps) are a class of membrane-associated proteins universally distributed among all bacteria. A characteristic N-terminal cysteine residue that is variably acylated anchors C-terminal globular domains to the extracellular surface, where they serve numerous roles, including in the capture and transport of essential nutrients. Lpps are also ligands for the Toll-like receptor 2 (TLR2) family, a key component of the innate immune system tasked with bacterial recognition. While Lpp function is conserved in all prokaryotes, structural heterogeneity in the N-terminal acylation state is widespread among Firmicutes and can differ between otherwise closely related species. In this study, we identify a novel two-gene system that directs the synthesis of N-acylated Lpps in the commensal and opportunistic pathogen subset of staphylococci. The two genes, which we have named the lipoprotein N-acylation transferase system (Lns), bear no resemblance to previously characterized N-terminal Lpp tailoring enzymes. LnsA (SAOUHSC_00822) is an NlpC/P60 superfamily enzyme, whereas LnsB (SAOHSC_02761) has remote homology to the CAAX protease and bacteriocin-processing enzyme (CPBP) family. Both LnsA and LnsB are together necessary and alone sufficient for N-acylation in Staphylococcus aureus and convert the Lpp chemotype from diacyl to triacyl when heterologously expressed in Listeria monocytogenes. Acquisition of lnsAB decreases TLR2-mediated detection of S. aureus by nearly 10-fold and shifts the activated TLR2 complex from TLR2/6 to TLR2/1. LnsAB thus has a dual role in attenuating TLR2 signaling in addition to a broader role in bacterial cell envelope physiology

Effects of Substitutions of Arginine Residues on the Basic Surface of Herpes Simplex Virus UL42 Support a Role for DNA Binding in Processive DNA Synthesis

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis

Resistance of Human Cytomegalovirus to the Benzimidazole l-Ribonucleoside Maribavir Maps to UL27

1-(β-d-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus

Inhibition of Human Cytomegalovirus Replication by Benzimidazole Nucleosides Involves Three Distinct Mechanisms

The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(β-l-ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs. We found that the erythrofuranosyl analog and the α-lyxofuranosyl analog acted late in the viral replication cycle, similar to BDCRB. In marked contrast, the α-5′-deoxylyxofuranosyl analog of 2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole (compound UMJD1311) acted early in the replication cycle, too early to be consistent with either mechanism. Similar to other reports on early acting inhibitors of herpesviruses, compound 1311 was multiplicity of infection dependent, an observation that could not be reproduced with UV-inactivated virus. HCMV isolates resistant to BDCRB and maribavir were sensitive to compound 1311, as were viruses resistant to ganciclovir, cidofovir, and foscarnet. The preincubation of host cells with compound 1311 and removal prior to the addition of HCMV did not produce an antiviral cellular response. We conclude that this newly discovered early mode of action occurs at a stage of viral replication after entry to cells but prior to viral DNA synthesis, thereby strongly suggesting that the trisubstituted benzimidazole nucleoside series possesses three distinct biochemical modes of action for inhibition of HCMV replication

Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus.

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains