Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease.

Despite significant advances in antiviral treatment, solid organ transplant (SOT) recipients remain at heightened risk for developing late cytomegalovirus (CMV) disease. Elevated inhibitory immune signaling suggests a state of immune impairment in SOT recipients, who do not control CMV infection and develop severe clinical symptoms after discontinuation of antiviral prophylaxis. We longitudinally monitored the negative immune modulator programmed death (PD)-1 receptor on both CD4 and CD8 T cells, co-expressing the CD137 surface marker of recent activation, in a liver transplant cohort. Liver recipients who progressed to CMV disease expressed elevated levels of PD-1 on CD137(+) CD4 and CD8 T cells, following stimulation with either full-length peptide libraries or CMV lysate. This novel approach, applicable to a multitude of human leukocyte antigen types, enhances the usefulness of the PD-1 measurements as a clinical strategy to predict late CMV disease. In parallel, we detected an increased level of the immunosuppressive cytokine interleukin (IL)-10, in plasma of liver recipients diagnosed with CMV disease. CMV-specific T cells were still functional when both PD-1 and IL-10 were upregulated; however they showed a marked proliferation deficit, which may limit their ability to contain viremia and lead to CMV disease. Our preliminary observations support further investigation of dual monitoring of PD-1 and IL-10, as potential immune markers of CMV disease.

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.

CD8+ T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indicating effects on virus gene expression.

Individuals infected with HIV-1 have varying rates of progression to AIDS. Cellular immune responses, comprised of cytolytic and noncytolytic CD8(+) T cell effector functions, are considered important for controlling viremia and maintaining the clinically asymptomatic state. Although there is general agreement regarding CD8(+) T lymphocyte cytotoxic functions, considerable controversy exists over the nature of the noncytolytic antiviral activity of CD8(+) cells. The discovery that RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1alpha, and MIP-1beta (macrophage inflammatory protein 1 alpha and beta) could inhibit HIV-1 replication by blocking viral entry processes led to the notion that these molecules are responsible for the CD8(+) cell suppressive activity. However, T tropic HIV isolates requiring the CXCR4 coreceptor for entry are insensitive to the antiviral effects of these beta-chemokines. Using a CXCR4-dependent virus, we determined that the mechanism of CD8(+) T cell-mediated activity did act after viral entry into the host cell. We also define the kinetics of the HIV life cycle in primary activated human CD4(+)-enriched T cells by using an HIV-1 reporter virus system pseudotyped with the CXCR4-dependent HIV-1 envelope gene of NL4-3. Analysis of these kinetic data indicates that CD8(+) T cell-mediated suppressive activity acts at a stage in the viral life cycle after entry and independently of the HIV envelope. Additionally, we show that the antiviral activity targets stages of the virus life cycle correlating with transcription and early proviral gene expression. These findings not only provide a range of possible targets for the CD8(+) T cell-mediated activity but also support the notion that this antiviral activity is multifactorial in nature.

Herpesvirus saimiri transformation of HIV type 1 suppressive CD8+ lymphocytes from an HIV type 1-infected asymptomatic individual.

CD8+ T lymphocytes from HIV+ individuals can potently suppress HIV-1 replication in a noncytolytic manner. This suppression appears to be multifactorial and the molecules contributing have not been fully elucidated. As an approach to this question we used herpesvirus saimiri (HVS) to transform CD8+ T lymphocytes from an HIV+ asymptomatic donor to a continuously growing, activation-independent, IL-2-dependent phenotype. The transformed cell population, termed CD8(HVS), had an activated phenotype, contained HVS sequences, did not shed infectious HVS virus, and was polyclonal. The CD8(HVS) cells, despite the absence of detectable CTL activity, potently suppressed HIV-1 production by both autologous and heterologous CD4+ cells from infected donors. The CD8(HVS) cells in coculture also suppressed virus production from PBMCs acutely infected with syncytium-inducing (SI) strains or NSI primary isolates of HIV-1. The supernatants from the CD8(HVS) cells and their concentrates derived from these supernatants were suppressive to NSI primary isolates of HIV-1 but not to SI strains. Fractionation of these concentrates showed that the suppressive activity was associated with low molecular mass (6500- to 19,300-Da) protein species. Western blotting and ELISA indicated that the CC chemokines MIP-1alpha, MIP-1beta, and RANTES were present in these fractions. Antibody-blocking studies with antibodies to the CC chemokines indicated that a significant portion of the soluble HIV-suppressive activity was due to these molecules. However, these experiments also suggested the inhibitory activity of the CD8(HVS) cells in coculture is not due exclusively to the CC chemokines. The HVS-transformed cells provide a useful tool for the study of noncytolytic CD8+ T lymphocyte-mediated suppression of HIV-1.

The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1.

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4(+) T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell-cell fusion, and examined levels of SDF-1 transcripts in CD8(+) T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the alpha and beta forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101-1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.

Novel mutation (V75T) in human immunodeficiency virus type 1 reverse transcriptase confers resistance to 2',3'-didehydro-2',3'-dideoxythymidine in cell culture.

We have selected a human immunodeficiency virus type 1 (HIV-1) mutant strain with a moderate (sevenfold) level of resistance to the nucleoside analog 2',3'-didehydro-2',3'-dideoxythymidine (D4T or stavudine). After serial passage of the HXB2 strain of HIV-1 in MT4 cells, a novel mutation involving two nucleotide substitutions in codon 75 of the viral reverse transcriptase, altering valine to threonine, was seen. When introduced into a wild-type HIV-1 background by site-directed mutagenesis, the T-75 mutation conferred cross-resistance to the dideoxynucleosides dideoxyinosine and dideoxycytosine as well as to 2',3'-didehydro-2',3'-dideoxycytosine.

Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance.

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.

Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine.

A series of biochemical investigations to compare the DNA polymerase and RNase H functions of the reverse transcriptases (RTs) corresponding to azidothymidine (AZT)-sensitive and -resistant human immunodeficiency virus (HIV) strains are described. Steady-state kinetic studies with purified recombinant enzymes utilizing several templates and three inhibitors, 3' azido-3' deoxythymidine triphosphate (AZTTP), 3-amino-thymidine 5'-triphosphate, and 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate, found consistent 2-4-fold differences between the enzymes from the two strains over a wide pH range. A strong pH dependence for all three inhibitors was found at pH values below 7.4 and suggested an ionizable group on the enzyme with a pK of about 7. The sensitivities of the RNase H activities of the two enzymes to AZTTP and AZTMP were also compared and found to be similar. The nucleotide incorporation fidelities of recombinant RTs corresponding to AZT-sensitive and -resistant clinical isolates were compared and the error specificities determined. No significant differences were found. Both enzymes were equally able to incorporate AZTTP into an elongating M13 DNA strand with concomitant chain termination. Purified wild-type and mutant virions from cell-culture supernatants were compared in "endogenous" DNA synthesis reactions, and the sensitivities of this activity to AZTTP were found to be similar. The contrast between the small differences found in this study and the high level of viral resistance in tissue culture presumably reflects an incomplete understanding of AZT inhibition of HIV in the cell.

Random mutagenesis of the thymidine kinase gene of varicella-zoster virus.

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260.

Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction.

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.

Structural analysis of Doc transposable elements associated with mutations at the white and suppressor of forked loci of Drosophila melanogaster.

DNA sequences from two spontaneous mutations of Drosophila melanogaster associated with insertion of a Doc transposable element have been cloned. In white-one, the element is inserted in the white locus close to where transcription initiates. In a lethal allele of suppressor of forked, su(f)S2, the element is inserted within the transcription unit in the protein coding region. Four other Doc elements have been cloned from a wild-type strain. Doc is a member of the class of transposable elements known as retroposons, which includes the D. melanogaster F, G, Jockey, and I elements. There is no sequence homology between the ends of the Doc element. The 3' or right end terminates with a polyadenylation signal sequence followed by a stretch of oligo-A. The length of the oligo-A varies between elements, and a duplication of variable size is found as a direct repeat flanking inserted Doc elements. Members of the family are conserved at the 3' end, but may be truncated at the 5' or left end. These structural features suggest a mechanism of transposition via an RNA intermediate.