Martha J. Lewis

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4− and CD8−, but CD44+, CD25+ and cytoplasmic CD3+. This phenotype is indicative of a thymus-derived pre–T-cell phenotype, and disease development was associated with the constitutive activation of NF-κB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Spontaneous production of C-C chemokines by individuals infected with human T lymphotropic virus type II (HTLV-II) alone and HTLV-II/HIV-1 coinfected individuals.

To investigate the immunological features of human T lymphotropic virus type II (HTLV-II) infection and specific mechanisms whereby HTLV-II might influence the progression of HIV-1 disease in coinfected individuals, we have analyzed the production of the C-C chemokines RANTES and macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) by PBMCs from HTLV-II-infected and HTLV-II/HIV-1-coinfected individuals. We observed spontaneous production of significant levels of MIP-1α and -1β and, to a lesser extent, RANTES, from individuals infected with HTLV-II alone or with concomitant HIV-1 infection. Spontaneous C-C chemokine production was not observed in PBMCs from uninfected or HIV-1-infected individuals. Although HTLV-II is known to preferentially infect CD8+ lymphocytes in vivo, we observed that whereas RANTES was produced exclusively by the CD8+-enriched fraction, MIP-1α and -1β were produced by both the CD8+-enriched and CD8+-depleted fractions of HTLV-II-infected PBMCs. RT-PCR demonstrated active expression of the HTLV-II regulatory protein Tax in the infected CD8+ T lymphocyte population, and it was further shown that Tax transactivates the promoters of MIP-1β and RANTES. Therefore, it appears that HTLV-II stimulates the production of C-C chemokines both directly at a transcriptional level via the viral transactivator Tax and also indirectly. Although the HTLV-II-infected individuals in this study are all virtually asymptomatic, they certainly display an abnormal immune phenotype. Moreover, our findings suggest that HTLV-II, via chemokine production, would be expected to alter the progression of HIV-1 infection in coinfected individuals.

Functional Adaptation of Nef to the Immune Milieu of HIV-1 Infection In Vivo

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4+ T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.

High rate of human T lymphotropic virus type IIa infection in HIV type 1-infected intravenous drug abusers in Ireland.

Serological and molecular analyses of a cohort of HIV-1-infected intravenous drug abusers (IVDAs) (n = 103) in Dublin, Ireland have demonstrated that 15 of 103 (14.6%) were infected with HTLV-II, which is the highest infection rate yet recorded for any European country. Restriction fragment length polymorphism (RFLP) analysis of the env region of the provirus demonstrated that the infection involved only the HTLV-IIa subtype; the HTLV-IIb subtype was not detected. Phylogenetic analysis of the nucleotide sequences of the long terminal repeat (LTR) confirmed infection with the HTLV-IIa subtype, and demonstrated that the viruses clustered closely with HTLV-IIa isolates from North American IVDAs. Previous observations that IVDAs in southern Europe, specifically Spain and Italy, appear to be infected predominantly with the HTLV-IIb subtype, along with the present report and evidence that IVDAs in Sweden are infected with the HTLV-IIa subtype, suggest different origins of HTLV-II infection in Europe.

Maintenance of Nef-Mediated Modulation of Major Histocompatibility Complex Class I and CD4 after Sexual Transmission of Human Immunodeficiency Virus Type 1

ABSTRACT Viruses encounter changing selective pressures during transmission between hosts, including host-specific immune responses and potentially varying functional demands on specific proteins. The human immunodeficiency virus type 1 Nef protein performs several functions potentially important for successful infection, including immune escape via down-regulation of class I major histocompatibility complex (MHC-I) and direct enhancement of viral infectivity and replication. Nef is also a major target of the host cytotoxic T-lymphocyte (CTL) response. To examine the impact of changing selective pressures on Nef functions following sexual transmission, we analyzed genetic and functional changes in nef clones from six transmission events. Phylogenetic analyses indicated that the diversity of nef was similar in both sources and acutely infected recipients, the patterns of selection across transmission were variable, and regions of Nef associated with distinct functions evolved similarly in sources and recipients. These results weighed against the selection of specific Nef functions by transmission or during acute infection. Measurement of Nef function provided no evidence that the down-regulation of either CD4 or MHC-I was optimized by transmission or during acute infection, although rare nef clones from sources that were impaired in these activities were not detected in recipients. Nef-specific CTL activity was detected as early as 3 weeks after infection and appeared to be an evolutionary force driving the diversification of nef. Despite the change in selective pressure between the source and recipient immune systems and concomitant genetic diversity, the majority of Nef proteins maintained robust abilities to down-regulate MHC-I and CD4. These data suggest that both functions are important for the successful establishment of infection in a new host.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo.

Functional analysis of human T lymphotropic virus type 2 Tax proteins

BackgroundThe Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays.ResultsWe found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis of the sequence of the two inactive Tax 2A proteins relative to Mo indicated that one had six amino acid changes and the other had one change in the central region of the protein. Mutations present at the amino and the extreme carboxy termini of Mo resulted in the loss of LTR but not NFkB activation whereas those occurring in the central region of the protein appeared to abolish transactivation of both promoters. Analysis of the transactivation phenotypes of Tax 1, Tax 2A Mo and Tax 2B containing mutations identified in the present study or previously characterised Tax mutations showed that domains required for LTR and NFkB activation are very similar but not identical in all three Tax proteins.ConclusionOur results suggest that loss of activity of two Tax 2A proteins derived from different isolates is associated with multiple amino acid changes relative to Mo in domains required for the activation of the CREB or CREB and NFkB pathways and that these domains are very similar but not identical in Tax 2B and Tax 1. The loss of Tax function in 2A viruses may have implications for their biological and pathogenic properties.

HIV-1 Quasispecies Delineation by Tag Linkage Deep Sequencing

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼0.005% to ∼0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.

Severe Acute Respiratory Syndrome Coronavirus Sequence Characteristics and Evolutionary Rate Estimate from Maximum Likelihood Analysis

In November 2002, a previously unknown severe acute respiratory syndrome (SARS) was observed in patients of the Guangdong Province, China (7). In March 2003, a new coronavirus (SARS-CoV) was associated with the SARS outbreak (2), and several full-genome sequences of SARS-CoV were obtained and compared (4). The family Coronaviridae comprises large, single, plus-stranded RNA viruses isolated from several species and previously known to cause common colds and diarrheal illnesses in humans (3). The emergence of such a novel, highly virulent pathogen warrants rapid investigation of its etiology and evolution to effectively control its impact on human health. In particular, estimating the rate of evolution of SARS-CoV would give an indication of how quickly the virus can potentially increase its genetic variability, which in turn has important implications for disease progression and drug and vaccine development. Phylogenetic analysis has proven successful for the investigation and prediction of the evolution of viruses such as influenza virus (1). Initial inspection of SARS-CoV sequences revealed a high degree of homogeneity, which might indicate an RNA virus that evolves unusually slowly. To investigate further, we carried out a full-genome alignment of the available SARS-CoV strains recently analyzed by Ruan et al. (4) by use of the CLUSTAL algorithm (6). The alignment was carefully edited by hand to maximize the number of identities, and the site positions containing gaps were removed. The resulting alignment (available from the authors upon request) is 21,333 nucleotides long; 63 sites have at least one sequence with a different nucleotide, and only 10 sites are phylogenetically informative, i.e., they are useful to discriminate among different tree topologies, according to the unweighted parsimony criterion. Subalignments were generated for all of the known coding regions, most of which were identical among the different isolates. We analyzed open reading frame (ORF) 1ab (4), which appears to be the most variable. Maximum likelihood (ML) methods were employed for the analyses because they allow for the testing of different phylogenetic hypotheses by calculating the probability of a given model of evolution generating the observed data and by comparing the probabilities of nested models by the likelihood ratio test (5). In addition, because only 10 sequences were retained after excluding the identical ones, it was possible to search for the optimal ML tree through an exhaustive or branch-and-bound search (5). Table ​Table11 shows the average base composition and the ML estimates of parameters describing the mode of evolution of SARS-CoV in ORF 1ab. The α parameter of the Γ distribution is extremely low (0.008), implying an extensive heterogeneity in the rate at which different nucleotide sites mutate along the genome. Moreover, the ML estimator implies that about 90% of the constant sites in the sequences are indeed invariable, i.e., they never change, possibly because of strong purifying selection. The variable sites, on the other hand, accumulate mutations very quickly. However, a note of caution is necessary because such a result may also be due to the small number of sequences available for analysis and the very short observation period. Table ​Table11 also shows that the hypothesis of a molecular clock cannot be rejected, although the P value is very close to 0.05; i.e., SARS-CoV isolates appear to be evolving at a constant evolutionary rate, which can be estimated from the ML tree with clock-like branch lengths shown in Fig. ​Fig.1.1. The branch lengths in the tree are proportional to the number of mutations accumulated by each viral lineage during evolution from the cenancestor, the most recent common ancestor. Assuming that the SARS-CoV cenancestor entered the human population 4 to 8 months ago (7), the evolutionary rate of the virus is of the order of 4 × 10−4 nucleotide changes per site per year (95% confidence interval [CI], 2.0 × 10−4 to 6 × 10−4) along the entire ORF 1ab. When only the variable sites are considered, the estimated rate is noticeably higher: 3.5 × 10−3 changes per site per year (95% CI, 2.6 × 10−3 to 4.4 × 10−3). This is the usual range for an RNA virus. Therefore, on average, eight point mutations are expected for the entire ORF 1ab region at each replication round. However, we cannot exclude the possibility that the sequence variability in the data sets is also affected by the passage of the virus in Vero cell culture before sequencing (4). Figure ​Figure11 also shows that the root of the tree, inferred by ML, is between the strains isolated from Hong Kong and Beijing, which are known to be epidemiologically linked to the strains isolated from patients in Guangdong Province and all the others (4). Epidemiological data also indicate that the index patient traveled from Guangdong to Hotel M in Hong Kong, where he transmitted the virus to several individuals who successively traveled to Singapore, Canada, and Vietnam (4). The tree shows, indeed, that the Singapore isolate and the isolates from Beijing belong to different, statistically supported clusters. However, because of the low phylogenetic signal, further classification of SARS-CoV isolates is not possible by phylogenetic analysis. All analyses confirm that SARS-CoV is not closely related to any known coronavirus (4), although it is assumed that the source must be one or more unidentified animal reservoirs in Asia. FIG. 1. Optimal ML tree of SARS-CoV ORF 1ab nucleotide sequences. Branch lengths are drawn proportional to the number of nucleotide changes per site and were estimated via ML enforcing a molecular clock and employing the HKY85+Γ+I nucleotide ... TABLE 1. Maximum likelihood estimators of nucleotide substitution model parameters for the SARS virus in ORF 1ab polyproteina In conclusion, the low sequence variability of SARS-CoV isolates is probably the consequence of its recent emergence in humans, but much greater viral heterogeneity with unpredictable consequences may be expected if the epidemic is not controlled. A rigorous phylogenetic approach might be an important tool to monitor the future evolution of the virus.

Partial Escape of HIV-1 From Cytotoxic T Lymphocytes During Chronic Infection

ABSTRACT Viral mutational escape from CD8+ cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or “partial escape.”