Characterization of HIV-2 chimeric viruses unable to use CCR5 and CXCR4 coreceptors.

We have previously shown the existence of primary human immunodeficiency virus type 2 isolates (MIC97 and MJC97) unable to use major coreceptors to entry into peripheral blood mononuclear cells, including CCR5 and CXCR4. We have now created a set of chimeric viruses derived from HIV-2(ROD), to study the contribution of env gene products in chemokine receptors usage and replication kinetics phenotype. The results obtained indicate that an env gene fragment, corresponding to the C1-C4 region of SU glycoprotein of both MIC97 and MJC97, impair efficient utilization of the major HIV coreceptors CCR5 and CXCR4 in phytohemagglutinin-stimulated peripheral blood mononuclear cells by ROD-MIC97 and ROD-MJC97 chimeric viruses. It also disrupts the ability to utilize established coreceptors for viral entry into GHOST-CD4 coreceptor-expressing cell lines. Resistance to CCR5 and CXCR4 inhibitors, as well as the ability to infect Delta32/Delta32ccr5 PBMC, was also observed in recombinant viruses containing the C1-C4 region from either MIC97 or MJC97. We also show that the presence of the TM region of env gene from MIC97 and MJC97 is sufficient to reduce viral replicative kinetics of ROD strain, indicating that this region, despite the presence and contribution of ROD genetic backbone, has an important role in viral progeny production efficiency.

Construction and characterization of CD4-independent infectious recombinant HIV-2 molecular clones.

Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) differ in their pathogenic mechanisms as evidenced by lower rate of disease progression, lower transmission rates and lower viral load in peripheral blood for HIV-2. One of the many factors that are involved in these characteristics is the interaction between viral glycoproteins and cellular receptors. The study of these interactions in an HIV-2 model could lead to important conclusions regarding pathogenesis and transmission mechanisms of HIV-2 infection. Here we report the design of a method enabling the construction of recombinant proviral HIV-2 DNAs in a moderate copy number plasmid that allows the analysis of env gene structure and functionality. This method constitutes an important tool for the study of HIV-2 env glycoproteins and for the mappings of genetic determinants of HIV-2 coreceptor usage and CD4-independent interaction. Furthermore, this knowledge will help towards the understanding of the different pathogenic mechanisms of HIV-1 and HIV-2.

Identification and characterization of HIV-2 strains obtained from asymptomatic patients that do not use CCR5 or CXCR4 coreceptors.

In vivo, human immunodeficiency virus type 2 (HIV-2) infection reveals several unique characteristics when compared to HIV-1 infection, the most remarkable of which is the extraordinarily long asymptomatic period. Here we describe two HIV-2 primary isolates, obtained from asymptomatic individuals, which do not infect any coreceptor-expressing cell lines tested. In those cells, we show that the absence of replication is directly related to cell entry events. Furthermore, productive infection observed in peripheral blood mononuclear cells (PBMC) was not inhibited by natural ligands and monoclonal antibodies directed to CCR5 and CXCR4. Finally, viral entry efficiency and viral progeny production of these viruses are markedly impaired in PBMC, indicating a reduced replicative fitness of both viruses. In conclusion, our data suggest that in some HIV-2 asymptomatic individuals, the circulating viruses are unable to use the major coreceptors to infect PBMC. This fact should have important implications in HIV-2 pathogenesis and transmission.

Discrimination of multidrug-resistant Mycobacterium tuberculosis IS6110 fingerprint subclusters by rpoB gene mutation analysis.

The rpoB gene mutations in a 69-bp region of the gene, resulting in resistance to rifampin, were used to discriminate between Mycobacterium tuberculosis IS6110 fingerprint subclusters. These subclusters exhibited identical IS6110 fragments or had one or two additional fragments. In the two major subclusters all the analyzed strains have the same variant rpoB allele but are different from each other, suggesting the occurrence of independent outbreaks.

Primary human immunodeficiency virus type 2 (HIV-2) isolates infect CD4-negative cells via CCR5 and CXCR4: comparison with HIV-1 and simian immunodeficiency virus and relevance to cell tropism in vivo.

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV(MAC)) can bind to and initiate infection of CD4(-) cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5(+) or CXCR4(+) cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and CXCR4 to infect CD4(+) cells were only able to use one of these receptors in the absence of CD4. Our observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo. Primary CD4(-) fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with soluble CD4. Thus, many primary HIV-2 strains infect CCR5(+) or CXCR4(+) cell lines without CD4 in vitro. CD4(-) cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation nonfunctional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect CD4(-) cells expressing CCR5 or CXCR4 in vivo.

Outbreak of multiple drug-resistant tuberculosis in Lisbon: detection by restriction fragment length polymorphism analysis.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) mainly among human immunodeficiency virus (HIV) seropositive patients in Lisbon hospitals in 1996-1997. OBJECTIVE: Detection of transmission of MDR-TB strains and epidemic outbreaks in several hospital units in the city of Lisbon, including a prison hospital. DESIGN: Use of restriction fragment length polymorphism (RFLP) to fingerprint isolates of Mycobacterium tuberculosis resistant to isoniazid, rifampicin, and one other drug. RESULTS: A total of 43 MDR-TB strains were typed. Sixty-seven per cent of the patients were HIV positive, 12% were HIV negative, and the remainder had unknown HIV status. About 88% of the isolates were grouped in three genetically similar clusters, suggesting possible recent transmission. A predominant cluster (cluster A), corresponding to 72% of the cases, was found, 45% of which came from the prison hospital. Strains from this cluster were resistant to isoniazid, rifampicin, streptomycin, and sometimes ethambutol. A retrospective epidemiological investigation was conducted with respect to all patients in cluster A, and epidemiological links were established between them. CONCLUSION: Our results suggest recent transmission of MDR-TB, mainly in HIV-positive patients, in Lisbon hospitals. Moreover, the predominant MDR-TB clustered strains were not confined to HIV-infected individuals, as they were also isolated in some immunocompetent patients.

Virological and molecular demonstration of human immunodeficiency virus type 2 vertical transmission.

To demonstrate that human immunodeficiency virus type 2 (HIV-2) mother-to-child transmission exists, HIV-2 isolates were obtained from both an asymptomatic mother (HIV-2 strain ARM), and her child (HIV-2 strain SAR), who had a diagnosis of AIDS. To determine their biological phenotype, primary isolates were used to infect various primary mononuclear cells and cell lines. HIV-2 ARM replicates in primary cells and Jurkat-tat, while HIV-2 SAR infects these cells plus SupT1, which led us to classify HIV-2 ARM as a slow/low virus and HIV-2 SAR as having an intermediate (slow/low-3) phenotype. Molecular analysis of the env region corresponding to gp125 was performed. Viral DNA was cloned, sequenced, and used to construct phylogenetic trees. The DNA sequence analysis demonstrated an overall nucleotide diversity of 7.6%. The results present evidence that the child's strain is more virulent than the mother's strain, which is in agreement with the immunodeficiency of the child. The phylogenetic trees that were constructed demonstrate that the two isolates cluster together, being closer to each other than to any other isolate described until now.

The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNA(Ala) gene of Mycobacterium spp.

Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5' end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3' end of the tRNA(Ala) gene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNA(Ala) gene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.

Insertion into the Mycobacterium smegmatis genome of the aph gene through lysogenization with the temperate mycobacteriophage Ms6.

A derivative of the temperate mycobacteriophage Ms6 containing the aph gene from transposon Tn5 was constructed. In the transductants the aph gene was integrated in the bacterial genome. The aph gene is stably maintained in the absence of positive selection after more than 150 generations. The results presented in this report show that Ms6 can be used as a vehicle for the integration of foreign DNA into the Mycobacterium smegmatis genome.

Structure of pAL5000, a plasmid from M. fortuitum and its utilization in transformation of mycobacteria.

We have developed a gene cloning system for mycobacteria. Based on the nucleotide sequence determined for the M. fortuitum plasmid pAL5000, we have constructed an E. coli/mycobacteria shuttle vector. This vector, pAL8, is composed of pAL5000, pTZ19R (an E. coli plasmid) and a kanamycin resistance gene (from Tn903). We were unable to obtain viable kanamycin resistant pAL8 transformants of M. smegmatis using a PEG-mediated DNA uptake system, in spite of the fact that we could show efficient DNA uptake by transfection using the mycobacterial lytic phage D29. However, kanamycin resistant transformants of M. smegmatis or BCG could be obtained by electroporation. This plasmid cloning system provides a tool for studies of the expression of cloned genes (e.g. virulence) or epitopes in mycobacteria and allows the rational construction of recombinant BCG polyvalent vaccines.

Restriction map of mycobacteriophage D29 and its deletion mutant F5.

A physical map of mycobacteriophage D29 was constructed, including positions for 25 restriction sites for 9 endunocleasic enzymes. D29 DNA contains about 48 150 bp. Analysis of a deletion mutant (F5) has allowed to determine the location of two non essential regions in the genome, allowing further insertion of foreign genes and construction of cosmids.

Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum.

We have determined the complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum; the plasmid contains 4837 bp with 65% G + C. Five open reading frames (ORF1 to ORF5) have been identified. A number of sequences corresponding to palindromes, repeats, a helix-turn-helix motif, a signal sequence and repetitive amino acid motifs can be identified. This sequence should facilitate the construction of vectors based on pAL5000 for transfer and expression studies in mycobacteria.

Effects of antituberculosis and antileprosy drugs on mycobacteriophage D29 growth.

The minimal inhibitory concentrations of antituberculosis and antileprosy drugs were determined for Mycobacterium aurum. The concentrations that reduced the final yield of bacteriophage D29R1 by 50% and the time during the replication cycle at which the drugs completely inhibited phage production were estimated. THe 50% inhibitory concentration/minimal inhibitory concentration ratios were close to 1.0 for clofazimine, colistin, rifampin, and streptomycin; these ratios were high for dapsone (diaminodiphenylsulfone) and isoniazid. Ethambutol (minimal inhibitory concentration, 1.0 micrograms/ml) was without effect on viral growth.