LTR Region Research Articles

Bifurcation and Enhancement of Autonomous-Nonautonomous Retrotransposon Partnership through LTR Swapping in Soybean

Long terminal repeat (LTR) retrotransposons, the most abundant genomic components in flowering plants, are classifiable into autonomous and nonautonomous elements based on their structural completeness and transposition capacity. It has been proposed that selection is the major force for maintaining sequence (e.g., LTR) conservation between nonautonomous elements and their autonomous counterparts. Here, we report the structural, evolutionary, and expression characterization of a giant retrovirus-like soybean (Glycine max) LTR retrotransposon family, SNARE. This family contains two autonomous subfamilies, SARE(A) and SARE(B), that appear to have evolved independently since the soybean genome tetraploidization event approximately 13 million years ago, and a nonautonomous subfamily, SNRE, that originated from SARE(A). Unexpectedly, a subset of the SNRE elements, which amplified from a single founding SNRE element within the last approximately 3 million years, have been dramatically homogenized with either SARE(A) or SARE(B) primarily in the LTR regions and bifurcated into distinct subgroups corresponding to the two autonomous subfamilies. We uncovered evidence of region-specific swapping of nonautonomous elements with autonomous elements that primarily generated various nonautonomous recombinants with LTR sequences from autonomous elements of different evolutionary lineages, thus revealing a molecular mechanism for the enhancement of preexisting partnership and the establishment of new partnership between autonomous and nonautonomous elements.

FISH analysis of a CRW-homology sequence from <I>Pseudoroegneria spicata</I> in <I>Thinopyrum ponticum</I> and <I>Th. intermedium</I>

To verify the genome components of Thinopyrum ponticum Liu alt; Wang and Th. intermedium [Host] Barkworth alt; Dewey, six specific primer pairs were designed according to the sequence of an autonomous centromeric retrotransposon of wheat (CRW) from Triticum boeoticum. Several DNA fragments were amplified by PCR from the genomic DNA of the diploid species Pseudoroegneria spicata A Löve. After sequence alignment, a 1.755 kb fragment was obtained and named as pStC1 (St genome centromere associated sequence, GenBank accession No. FJ952565). This fragment contained an 800 bp fragment highly homologous to the LTR region of autonomous CRW, a short fragment partial homology to the gag region of CRW, and an AGCAAC-rich tandem repeat. Fluorescent in situ hybridization (FISH) using pStC1 as a probe was carried out on the chromosomes of Th. ponticum, Th. intermedium and Triticum aestivum cv. Chinese Spring. Th. ponticum was proved to have two St and three E genomes (St1St2EeEbEx). In Th. intermedium, strong FISH signals were observed on St genome chromosomes, while faint signals were also found on some E-genome chromosomes at their pericentromeric regions. These results indicated that during speciation of sub-genomes in the allopolyploids of Thinopyrum genus, concerted evolution might have occurred at centromeric and pericentromeric regions.

Viral Genetic Determinants of Nonprogressive HIV Type 1 Subtype C Infection in Antiretroviral Drug-Naive Children

Characterization of HIV-1 from slow progressors is important to facilitate vaccine and antiviral drug development. To identify virus attenuations that may contribute to slower rates of disease progression, the full-length viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Proviral DNA was extracted from cocultured peripheral blood mononuclear cells and used to PCR amplify, sequence, and extensively analyze the near full-length genomes and LTR regions. All primary HIV-1 isolates were HIV-1 subtype C throughout their genome, and amino acid (AA) sequence analysis revealed open reading frames for all genes. However, all isolates had at least one unusual gene/protein. For example, isolate LT5 had a 2AA insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5AA in the C-terminus of tat exon 2, while integrase in isolate LT39 had an additional 4AA at the C-terminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16AA truncation, and in addition, had a further 3AA. Furthermore, altered functional domains were noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreased proapoptotic activity (all isolates), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). The slower HIV-1 disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore is inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

Caracterização molecular do HTLV-1/2 em doadores de sangue em Belém, Estado do Pará: primeira descrição do subtipo HTLV-2b na região Amazônica

This study aimed to perform molecular characterization on the human T-cell lymphotropic virus (HTLV) infecting blood donors attended at the Hematology and Hemotherapy Center-Foundation of Pará. DNA samples from 79 HTLV-seropositive individuals were analyzed by means of the polymerase chain reaction on the pX, env and 5'LTR genomic regions; restriction fragment length polymorphism analysis; and sequencing of the 5'LTR region with subsequent phylogenetic analysis. From this, the HTLV types and subtypes circulating in the study population were defined. There was higher prevalence of HTLV-1 (71%) than of HTLV-2 (29%). HTLV-1 samples were classified as belonging to the Cosmopolitan subtype, Transcontinental subgroup; and the HTLV-2 samples as HTLV-2c. Analysis on the restriction fragment length polymorphisms of the env region and sequencing of the 5'LTR region identified a sample of HTLV-2b, for the first time in the Brazilian Amazon region. This emphasizes the need for ongoing molecular studies in this region, in order to have better understanding of the epidemiology of HTLV transmission in the population, and to enable epidemiological surveillance of the emergence of new types and subtypes.

Divergent Strains of Human T-Lymphotropic Virus Type 1 (HTLV-1) within the Cosmopolitan Subtype in Argentina

HTLV-1 Cosmopolitan subtype Transcontinental subgroup A has been described among aboriginal communities from the northwest endemic area of Argentina. Moreover, Transcontinental subgroup A and the Japanese subgroup B were reported among blood donors from the nonendemic central region of the country. We carried out the first HTLV-1 phylogenetic study in individuals residing in Buenos Aires capital city. Phylogenetic analysis performed on the LTR region showed that all 44 new strains clustered within the Cosmopolitan subtype, with 42 (95.4%) belonging to Transcontinental subgroup A. Of them, 20 (45.5%) strains grouped in the large Latin American cluster and 4 (9.1%) in the small Latin American cluster. The majority of them belonged to individuals of nonblack origin, grouped with Amerindian strains. Three (6.8%) were closely related to South African references and two monophyletic clusters including only HIV/HTLV-1 coinfected individuals were observed. Interestingly, two (4.5%) new sequences (divergent strains) branched off from all five known Cosmopolitan subgroups in a well-supported clade. In summary, these findings show that HTLV-1 Cosmopolitan subtype Transcontinental subgroup A is infecting residents of Buenos Aires, a nonendemic area of Argentina, and confirm the introduction of divergent strains in the country.

HTLV‐1 infection in blood donors from the Western Brazilian Amazon region: Seroprevalence and molecular study of viral isolates

To determine the seroprevalence of HTLV-1 in Brazil, and to review the virus molecular epidemiology in this Amazon population (Rio Branco-Acre), 219 blood donors were screened for HTLV-1. Only one case of infection (0.46% seroprevalence) was detected during July 2004 screening at the Acre Hospital Foundation (FUNDACRE). Neighbor-joining and Maximum Likelihood phylogenetic analyses of two (n = 2) complete LTR region sequences were performed with the PAUP* software. Since the HTLV-1 envelope surface (gp46) and transmembrane (gp21) glycoproteins are important for virus fitness, three envelope glycoproteins sequences (n = 3) were analyzed using the Prosite tool to determinate potential protein sites. Phylogenetic analysis demonstrated that the new isolate described in this study, and the unpublished LTR strain described in a previous report belong to the Transcontinental subgroup of the Cosmopolitan subtype, inside the Latin American cluster. A similar result was obtained when submitting, to the Automated Genotyping System, three LTR partial sequences from a previous study of the seroprevalence of HTLV-1 in the same Amazon population. In all analyzed env sequences, the potential protein site was found: two PKC phosphorylation sites at amino acid (aa) positions 310-312 and 342-344, one CK2 phosphorylation site at 194-197aa, three N-glycosylation sites at 222-225aa, 244-247aa and 272-275aa, and a single N-myristylation site at 327-338aa. In conclusion, potential protein sites described in HTLV-1 gp46 and gp21 confirm the presence of conserved sites in the HTLV-1 envelope proteins, likewise phylogenetic analysis suggests a possible recent introduction of the virus into North Brazil.

Phylogenetic and molecular analysis of HTLV‐1 isolates from a medium sized town in Northern of Brazil: Tracing a common origin of the virus from the most endemic city in the country

Salvador-Bahia has the highest prevalence of HTLV-1 infection in Brazil; about 2% of the population is infected. In this city, the prevalence of HTLV in pregnant women is 1%. There is no data of the HTLV-1 prevalence in others cities of the Bahia's Recôncavo, where the population has similar social and demography characteristics to those from Salvador. Our aim was to evaluate the seroprevalence of HTLV in pregnant women in Cruz das Almas-Bahia, a medium-sized city from the Bahia's Recôncavo. All individuals were tested for HTLV (ELISA) and the positive samples were confirmed by Western Blot. Phylogenetic analyses of the total LTR region were performed in all positive samples. We tested 408 samples (45.4% of the estimate pregnant women population) between June 1st and October 31, 2005. The prevalence of HTLV-1 infection was 0.98%. In addition, all isolated virus were grouped in the subtype HTLV-1a, in the Latin American group. Our results suggest that the introduction of HTLV-1 occurred after the slave trade into Salvador. In addition, HTLV-1-infection should be screened during the pregnancy in women originating from HTLV-1 endemic areas.

HTLV Type 1 Genetic Types among Native Descendants in Argentina

The province of San Salvador de Jujuy, located in the northwest of Argentina, is a highly endemic area for HTLV-1 infection and a foci of tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP). Therefore, to better understand this, we carried out a genetic characterization of a large set of HTLV-1 strains (n = 65) of descendants of Amerindians from this region. The LTR and env regions were analyzed. The genetic analysis showed that all of these new HTLV-1 isolates from Argentina belong to the Transcontinental subgroup A of the HTLV-1a Cosmopolitan subtype, with the exception of three isolates that cluster within the Japanese subgroup B. Interestingly, the majority of the sequences from Jujuy province belonged to a distinct cluster within the Latin America Transcontinental subgroup, referred to here as the Jujuy subcluster, and were characterized by specific signatures in the LTR. Given that the samples analyzed in this study belong to the Amerindian population and the high prevalence of HTLV-1 in Jujuy in contrast to the low prevalence of this virus in the country, it could be that HTLV-1aA was spread in Argentina from the Amerindians to the cosmopolitan population. Moreover, this is the first report of an HTLV-1aB or Japanese subgroup in descendants of non-Japanese people in South America.

APOBEC3G-independent reduction in virion infectivity during long-term HIV-1 replication in terminally differentiated macrophages

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. In the case of HIV, the antiviral activity of APO3G is counteracted by the viral Vif protein. Monocyte-derived macrophages (MDM) are terminally differentiated, non-dividing cells susceptible to HIV infection. Human MDM are known to express APO3G and HIV replication in these cells is dependent on Vif. Here we analyzed the correlation between HIV-1 replication and APO3G expression in MDM. Replication of wild type HIV-1 induced a gradual 4–5-fold reduction in APO3G expression. The efficiency of APO3G downregulation correlated with the efficiency of virus replication. Interestingly, despite downregulation of APO3G, the relative infectivity of viruses rapidly declined during the course of infection and was already reduced ∼ 90% prior to peak virus production. Cell-free virus preparations showed increased levels of a 41 kDa MA–CA processing intermediate. Sequence analysis around the MA–CA cleavage site and the protease and LTR regions did not reveal deaminase-induced hypermutation of the viral genome, suggesting that APO3G activity is not responsible for the incomplete Gag processing. Thus, the loss of infectivity of HIV-1 viruses produced from long-term infected primary macrophages is due to an APO3G-independent mechanism.

Invasion of the Arabidopsis Genome by the Tobacco Retrotransposon Tnt1 Is Controlled by Reversible Transcriptional Gene Silencing

Long terminal repeat (LTR) retrotransposons are generally silent in plant genomes. However, they often constitute a large proportion of repeated sequences in plants. This suggests that their silencing is set up after a certain copy number is reached and/or that it can be released in some circumstances. We introduced the tobacco (Nicotiana tabacum) LTR retrotransposon Tnt1 into Arabidopsis (Arabidopsis thaliana), thus mimicking the horizontal transfer of a retrotransposon into a new host species and allowing us to study the regulatory mechanisms controlling its amplification. Tnt1 is transcriptionally silenced in Arabidopsis in a copy number-dependent manner. This silencing is associated with 24-nucleotide short-interfering RNAs targeting the promoter localized in the LTR region and with the non-CG site methylation of these sequences. Consequently, the silencing of Tnt1 is not released in methyltransferase1 mutants, in contrast to decrease in DNA methylation1 or polymerase IVa mutants. Stable reversion of Tnt1 silencing is obtained when the number of Tnt1 elements is reduced to two by genetic segregation. Our results support a model in which Tnt1 silencing in Arabidopsis occurs via an RNA-directed DNA methylation process. We further show that silencing can be partially overcome by some stresses.

HTLV Type 1 Molecular Study in Brazilian Villages with African Characteristics Giving Support to the Post-Columbian Introduction Hypothesis

We performed an HTLV epidemiological study of 986 individuals from 17 villages from the same state of Salvador, the city with the highest HTLV-1 prevalence in Brazil. The HTLV-1 prevalence was 3.85%, 1.56%, and 1.23% in three villages. Phylogenetic analysis of the LTR region demonstrated that all positive samples analyzed belonged to the Transcontinental subgroup of the HTLV-1 Cosmopolitan subtype. Three of the new HTLV-1 sequences formed a well-supported clade within one of the Latin American clusters that contain a South African sequence. This Latin American cluster that segregated from the same ancestor as the other clade contained a Central African sequence. This ancestral relationship could support our previous report that suggests that this subgroup was first introduced into South Africa as a result of the migration of the Bantu population from Central Africa to Southern Africa over the past 3000 years, and afterward to Brazil during the slave trade between the sixteenth and nineteenth centuries.

High Frequency of Gross Deletions in the 5′ LTR andgagRegions in HIV Type 1-Infected Long-Term Survivors Treated with Korean Red Ginseng

Our previous studies have shown that gross deletions in the nef gene as well as slow decreases in CD4 T cell numbers are associated with Korean red ginseng (KRG) intake in HIV-1-infected patients. To determine whether there might be an association between KRG intake and occurrence of gross deletions (gDelta) in the 5' LTR and gag regions, we examined the 1125 base pair (bp) sequences encompassing these regions in 10 long-term survivors (LTSs) treated with KRG (total of 13,364 +/- 5364 g) for > 12 years, and in 8 LTS control patients with no or minimal (total of 1436 +/- 1027 g) KRG intake (LTS controls). In the 10 LTSs, 189 PCR products were obtained from 80 peripheral blood mononuclear cell (PBMC) samples. In total, 44 of the 80 PBMC samples (55%) and 71 of the 189 PCR products (37.6%) displayed gDelta. While 55% of PBMC samples and 37.6% of PCR products showed gDelta in the 10 LTSs, the corresponding figures for the eight LTS controls were 30.3% and 14.8%. These differences were significant (p < 0.05 and p = 0, respectively). In addition, the proportions of 28 patients in the general population (without KRG intake) displaying PBMC and PCR gDelta were 13.3% and 8.3%, respectively. Our data strongly suggest that gDelta occurrence in the HIV-1 5' LTR and gag regions is associated with KRG intake.

Genomic organization, sequence divergence, and recombination of feline immunodeficiency virus from lions in the wild.

BackgroundFeline immunodeficiency virus (FIV) naturally infects multiple species of cat and is related to human immunodeficiency virus in humans. FIV infection causes AIDS-like disease and mortality in the domestic cat (Felis catus) and serves as a natural model for HIV infection in humans. In African lions (Panthera leo) and other exotic felid species, disease etiology introduced by FIV infection are less clear, but recent studies indicate that FIV causes moderate to severe CD4 depletion.ResultsIn this study, comparative genomic methods are used to evaluate the full proviral genome of two geographically distinct FIV subtypes isolated from free-ranging lions. Genome organization of FIVPle subtype B (9891 bp) from lions in the Serengeti National Park in Tanzania and FIVPle subtype E (9899 bp) isolated from lions in the Okavango Delta in Botswana, both resemble FIV genome sequence from puma, Pallas cat and domestic cat across 5' LTR, gag, pol, vif, orfA, env, rev and 3'LTR regions. Comparative analyses of available full-length FIV consisting of subtypes A, B and C from FIVFca, Pallas cat FIVOma and two puma FIVPco subtypes A and B recapitulate the species-specific monophyly of FIV marked by high levels of genetic diversity both within and between species. Across all FIVPle gene regions except env, lion subtypes B and E are monophyletic, and marginally more similar to Pallas cat FIVOma than to other FIV. Sequence analyses indicate the SU and TM regions of env vary substantially between subtypes, with FIVPle subtype E more related to domestic cat FIVFca than to FIVPle subtype B and FIVOma likely reflecting recombination between strains in the wild.ConclusionThis study demonstrates the necessity of whole-genome analysis to complement population/gene-based studies, which are of limited utility in uncovering complex events such as recombination that may lead to functional differences in virulence and pathogenicity. These full-length lion lentiviruses are integral to the advancement of comparative genomics of human pathogens, as well as emerging disease in wild populations of endangered species.

Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53

The evolutionary forces that establish and hone target gene networks of transcription factors are largely unknown. Transposition of retroelements may play a role, but its global importance, beyond a few well described examples for isolated genes, is not clear. We report that LTR class I endogenous retrovirus (ERV) retroelements impact considerably the transcriptional network of human tumor suppressor protein p53. A total of 1,509 of approximately 319,000 human ERV LTR regions have a near-perfect p53 DNA binding site. The LTR10 and MER61 families are particularly enriched for copies with a p53 site. These ERV families are primate-specific and transposed actively near the time when the New World and Old World monkey lineages split. Other mammalian species lack these p53 response elements. Analysis of published genomewide ChIP data for p53 indicates that more than one-third of identified p53 binding sites are accounted for by ERV copies with a p53 site. ChIP and expression studies for individual genes indicate that human ERV p53 sites are likely part of the p53 transcriptional program and direct regulation of p53 target genes. These results demonstrate how retroelements can significantly shape the regulatory network of a transcription factor in a species-specific manner.

The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.

The Close Relationship between South African and Latin American HTLV Type 1 Strains Corroborated in a Molecular Epidemiological Study of the HTLV Type 1 Isolates from a Blood Donor Cohort

It has been difficult to explain why all HTLV-1 sequences in Salvador, a city in the northeast of Brazil, belong to the Transcontinental (A) subgroup of the Cosmopolitan (a) subtype, since according to historical data the vast majority of slaves brought to Brazil (through Salvador) came from west Africa, where only the western African subgroup (C) has been found. To shed more light on this subject we conducted a phylogenetic analysis of 23 isolates from blood donors of Salvador. DNA was extracted and submitted to a nested PCR for amplification of the entire LTR region. The PCR products were purified and sequenced on an automated sequencer. Neighbor-joining and maximum likelihood phylogenetic analyses were performed. None of the new sequences from Salvador clustered within the West-African subgroup C. Confirming previous results, all sequences belonged to the Transcontinental subgroup (A) of the Cosmopolitan subtype, and clustered in two Latin American clusters. In addition we showed sequences from southern Africa clustering in both Latin American clusters. One of the new sequences is ancestral to the larger Latin American cluster beta due to a duplication of a 12-bp long fragment, a finding that has not been previously described. These findings support the hypothesis that HTLV-1 isolates circulating in Latin America have a closer relationship to South African compared to West-African HTLV-1 strains. The 12-bp-long duplications in one of the sequences has no obvious clinical or biological implications yet.

Distribution of Human T Cell Lymphotropic Virus Type 1 (HTLV-1) Subtypes in Brazil: Genetic Characterization of LTR and Tax Region

We report the molecular and epidemiological characterization of 128 human T cell lymphotropic virus type 1 (HTLV-1) isolates from Brazilian patients with different clinical manifestations of the infection. Thirty-two percent of the patients were asymptomatic, 44% had HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 23% had adult T cell leukemia/lymphoma (ATLL). Phylogenetic analysis performed using part of the LTR region of the viral genome revealed that all Brazilian isolates belonged to the Cosmopolitan subtype, with the following distribution within the Transcontinental subgroup: 81.6% within the Latin American cluster and 15.8% outside the Latin American cluster. Two isolates belonged to the Japanese subgroup. Molecular analysis of the tax region showed a high nucleotide similarity ( approximately 99%) with 41 prototype sequences, including the ATK-1 isolate. The mean number of nucleotide substitutions ranged from 1 to 8. Five specific nucleotide substitutions, C7401T, T7914C, C7920T, C7982T, and G8231A, were highly conserved among the Brazilian isolates (79.6%), with a frequency ranging from 81.6% to 100% in the sample group and from 18.4% to 24.1% in the prototypes used, suggesting the existence of a molecular signature. These changes were not correlated with a specific clinical status of the patients and could be a molecular characteristic of the HTLV-1 strains that circulate in Brazil.

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions ( pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12–26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.

Genetic characterization of human immunodeficiency virus type 1 from Beira, Mozambique

In this report, we examined the genetic diversity of HIV-1 strains circulating in the city of Beira, the second largest metropolitan area in Mozambique. A total of 131 blood samples, collected between August and October 2003 from antiretroviral-naïve individuals, were characterized with a combined approach consisting of heteroduplex mobility assay (HMA) subtyping for gag ( n = 74) and/or env ( n = 117) genes, and DNA sequence analysis of proviral env (C2V3C3, n = 52), LTR ( n = 30) and/or pol ( n = 43) genomic regions. Aside from the identification, by bootscanning analysis, of a viral strain with a C/A 1 mosaic C2V3C3 structure, classified as subtype A by env HMA, phylogenetic inference studies of the sequence data demonstrated the circulation of genetically diverse subtype C viruses, predominantly of the R5 type. Inspection of the LTR sequences revealed a pattern of structural and regulatory elements typical of subtype C, with 63.3% of the viruses showing three NF-κB binding sites. Analysis of the predicted protease sequences enabled us to detect a single primary mutation (I84V, n = 1) associated with resistance to protease inhibitors (PI), while secondary mutations were highly prevalent, some of them in combinations which may confer PI resistance. Although an unexpectedly high rate (11.6%) of reverse transcriptase key mutations (V75A, K103N, Y181C, M184I, or P236L) was detected in the sequences analyzed, our data suggest the non-epidemic circulation of resistant viruses, and the absence of multi-class drug resistant viral strains.

Retroelement Genome Painting: Cytological Visualization of Retroelement Expansions in the Genera Zea and Tripsacum

Divergence of abundant genomic elements among the Zea and Tripsacum genera was examined cytologically and a tool kit established for subsequent studies. The LTR regions from the CRM, Huck, Grande, Prem1, Prem2/Ji, Opie, Cinful-1, and Tekay retroelement families were used as FISH probes on mitotic chromosome spreads from a "trispecies" hybrid containing chromosomes from each of three species: Zea mays (2n = 20), Z. diploperennis (2n = 20), and Tripsacum dactyloides (2n = 36). Except for Tekay, which painted both Zea and Tripsacum chromosomes with nearly equal intensity, the retroelement probes hybridized strongly to the Zea chromosomes, allowing them to be distinguished from those of Tripsacum. Huck and Grande hybridized more intensely to maize than to Z. diploperennis chromosomes. Tripsacum genomic clones containing retroelement sequences were isolated that specifically paint Tripsacum chromosomes. The retroelement paints proved effective for distinguishing different genomes in interspecific hybrids and visualizing alien chromatin from T. dactyloides introgressed into maize lines. Other FISH probes (180-bp knob, TR-1, 5S, NOR, Cent4, CentC, rp1, rp3, and alpha-ZeinA) could be simultaneously visualized with the retroelement probes, emphasizing the value of the retroelement probes for cytogenetic studies of Zea and Tripsacum.