Resistance of human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps to UL27

1-(beta-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-(beta-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.     

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection

Background  

Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.     

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.     

Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.     

Mutations of the woodchuck hepatitis virus polymerase gene that confer resistance to lamivudine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil.

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression.

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.     

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.     

Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine.

Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug.     

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.     

Accelerated evolution of maribavir resistance in a cytomegalovirus exonuclease domain II mutant

A human cytomegalovirus (CMV) UL54 pol exonuclease domain II mutation, D413A, found in a clinical specimen, conferred ganciclovir (GCV) and cidofovir resistance but not foscarnet resistance when incorporated into laboratory strain T2294. After several passages without drug, mutation was observed in five of eight plaques of T2294, and its plating efficiency under foscarnet was increased ~30-fold over that of a control strain. When T2294 was serially passed under maribavir (MBV), phenotypic changes and viral UL97 mutations were detected by passage 5, much earlier than previously reported for other CMV strains. By passage 15, mutations included two cases of H411Y, one each of H411L and H411N, three of T409M, five of V353A, and one of L397R. Five instances of codon 409 or 411 mutations evolved into double mutations including V353A. Marker transfer experiments showed H411N/Y/L to confer 9- to 70-fold-increased MBV resistance and combinations of H411L/Y and V353A to confer >150-fold-increased MBV resistance, but no GCV resistance. These findings are consistent with defective exonuclease activity of the pol D413A mutant T2294, leading to the accelerated evolution of UL97 mutations under MBV. This recapitulated the known resistance mutations V353A, L397R, and T409M; suggested their relative frequency; and identified new ones at codon 411. These UL97 mutations predict an MBV binding region overlapping the kinase ATP binding site and located upstream of known GCV resistance mutations. The existence of viable pol D413A mutants may facilitate the selection of additional drug resistance mutations in vivo and the study of these mutations in vitro.     

Rapid detection of human cytomegalovirus UL97 and UL54 mutations for antiviral resistance in clinical specimens

Drug‐resistant cytomegalovirus appears during prolonged anti‐cytomegalovirus therapy. Assays for human cytomegalovirus viral protein kinase (UL97) and viral DNA polymerase (UL54) gene mutations conferring drug resistance have been used rather than susceptibility assays to assess clinical specimens. In this study a sensitive system for genotype assay of UL97 and UL54 in clinical specimens with as few as six copies/µg of DNA was developed.     

The UL16 gene of human cytomegalovirus encodes a glycoprotein that is dispensable for growth in vitro.

The UL16 gene of human cytomegalovirus (HCMV) encodes a predicted translation product with features characteristic of glycoproteins (signal and anchor sequences and eight potential N-linked glycosylation sites). Antisera were raised against the UL16 gene product expressed in Escherichia coli as a beta-galactosidase fusion protein. The antisera detected a 50-kDa glycoprotein in HCMV-infected cells that was absent from purified virions. The UL16 glycoprotein was synthesized at early times after infection and accumulated to the highest levels at late times after infection. A recombinant HCMV in which UL16 coding sequences were interrupted by a lacZ expression cassette was constructed by insertional mutagenesis. Analysis of the phenotype of the recombinant virus indicated that the UL16 gene product is nonessential for virus infectivity and growth in tissue culture.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Mutations that confer resistance to broadly-neutralizing antibodies define HIV-1 variants of transmitting mothers from that of non-transmitting mothers

Despite considerable reduction of mother-to-child transmission (MTCT) of HIV through use of maternal and infant antiretroviral therapy (ART), over 150,000 infants continue to become infected with HIV annually, falling far short of the World Health Organization goal of reaching <20,000 annual pediatric HIV cases worldwide by 2020. Prior to the widespread use of ART in the setting of pregnancy, over half of infants born to HIV-infected mothers were protected against HIV acquisition. Yet, the role of maternal immune factors in this protection against vertical transmission is still unclear, hampering the development of synergistic strategies to further reduce MTCT. It has been established that infant transmitted/founder (T/F) viruses are often resistant to maternal plasma, yet it is unknown if the neutralization resistance profile of circulating viruses predicts the maternal risk of transmission to her infant. In this study, we amplified HIV-1 envelope genes (env) by single genome amplification and produced representative Env variants from plasma of 19 non-transmitting mothers from the U.S. Women Infant Transmission Study (WITS), enrolled in the pre-ART era. Maternal HIV Env variants from non-transmitting mothers had similar sensitivity to autologous plasma as observed for non-transmitting variants from transmitting mothers. In contrast, infant variants were on average 30% less sensitive to paired plasma neutralization compared to non-transmitted maternal variants from both transmitting and non-transmitting mothers (p = 0.015). Importantly, a signature sequence analysis revealed that motifs enriched in env sequences from transmitting mothers were associated with broadly neutralizing antibody (bnAb) resistance. Altogether, our findings suggest that circulating maternal virus resistance to bnAb-mediated neutralization, but not autologous plasma neutralization, near the time of delivery, predicts increased MTCT risk. These results caution that enhancement of maternal plasma neutralization through passive or active vaccination during pregnancy may potentially drive the evolution of variants fit for vertical transmission.     

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.