The AG recombinant IbNG and novel strains of group M HIV-1 are common in Cameroon.

The genetic diversity of group M HIV-1 is highest in west central Africa. Blood samples from four locations in Cameroon were collected to determine the molecular epidemiology of HIV-1. The C2-V5 region of envelope was sequenced from 39 of the 40 samples collected, and 7 samples were sequenced across the genome. All strains belonged to group M of HIV-1. The circulating recombinant form CRF02 AG (IbNG) was the most common strain (22/39, 56%). Two of these were confirmed by full genome analysis. Four samples (4/39, 10%) clustered with the sub-subtype F2 and one of these was confirmed by full genome sequencing. Recombinant forms, each different but containing subtype A, accounted for the next most common form (7/39, 18%). Among these recombinants, those combining subtypes A and G were the most common (4/7, 57%). Also found were 3 subtype A, 2 subtype G, and 1 subtype B strain. Many recombination break points were shared between IbNG and the other AG recombinants, though none of these other AG recombinants included IbNG as a parent. This suggests that there was an ancestral AG recombinant that gave rise to CRF02 AG (IbNG), the successful circulating recombinant form, and to others that were less successful and are now rare.

Clinical implications of identifying non-B subtypes of human immunodeficiency virus type 1 infection.

Although human immunodeficiency virus type 1 (HIV-1) infection in the United States has predominantly involved subtype B, increasing global travel is leading to wider dissemination of genetically heterogeneous subtypes. While physicians depend on HIV-1 viral load measurements to guide antiretroviral therapy, commonly used molecular assays may underestimate the viral load of patients with non-B subtypes. Nine patients with non-B subtypes of HIV-1 were identified by physicians who suspected a non-B subtype on the basis of a low or undetectable HIV-1 viral load, by the Amplicor HIV-1 Monitor test, version 1.0, in conjunction with either a declining CD4 cell count or history of travel outside the United States. Use of version 1.5 of the Amplicor HIV-1 Monitor test detected a median HIV-1 viral load that was 2.0 log(10) RNA copies/mL higher than was determined with version 1.0. Clinical management was altered in all cases after diagnosis of a non-B-subtype infection. These cases demonstrate that it is critical for physicians to suspect and diagnose non-B subtypes of HIV-1 so that an assay with reliable subtype performance can be used to guide antiretroviral therapy.

Evaluation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay using primary subtype A, C, and D isolates from Kenya.

Accurate and sensitive quantification of human immunodeficiency virus type 1 (HIV-1) RNA has been invaluable as a marker for disease prognosis and for clinical monitoring of HIV-1 disease. The first generation of commercially available HIV-1 RNA tests were optimized to detect the predominant HIV-1 subtype found in North America and Europe, subtype B. However, these tests are frequently suboptimal in detecting HIV-1 genetic forms or subtypes found in other parts of the world. The goal of the present study was to evaluate the performance of a new viral load assay with non-subtype B viruses. A transcription-mediated amplification method for detection and quantitation of diverse HIV-1 subtypes, called the Gen-Probe HIV-1 viral load assay, is under development. In this study we examined the performance of the Gen-Probe HIV-1 viral load assay relative to that of the commonly used commercial HIV-1 RNA assays using a panel of primary isolates from Kenya. For comparison, we included several subtype B cloned viruses, and we quantified each virus using an in-house quantitative-competitive reverse transcriptase PCR (QC-RT-PCR) method and gag(p24) antigen capture. The Gen-Probe HIV-1 viral load assay and a version of the Roche AMPLICOR HIV-1 MONITOR test (version 1.5) that was designed to detect a broader range of subtypes were both sensitive for the quantification of Kenyan primary isolates, which represented subtype A, C, and D viruses. The Gen-Probe HIV-1 viral load assay was more sensitive for the majority of viruses than the Roche AMPLICOR HIV-1 MONITOR test version 1.0, the Bayer Quantiplex HIV RNA 3.0 assay, or a QC-RT-PCR method in use in our laboratory, suggesting that it provides a useful method for quantifying HIV-1 RNAs from diverse parts of the world, including Africa.

Use of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 to assess the performance of viral RNA quantitation tests.

Optimal management of human immunodeficiency virus type 1 (HIV-1) disease requires accurate quantitation of viral RNA concentrations in plasma. Evidence for increasing geographic intermixing of HIV-1 subtypes makes equivalent quantitation of all subtypes essential. The performances of six quantitative viral RNA tests are described, for the first time, with calibrated viral isolates of diverse subtypes.

Development of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 and performance of an improved AMPLICOR HIV-1 MONITOR test with isolates of diverse subtypes.

Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.

Antiviral effect and ex vivo CD4+ T cell proliferation in HIV-positive patients as a result of CD28 costimulation.

Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.

Identification of the human herpesvirus 6 glycoprotein H and putative large tegument protein genes.

Determination of the nucleotide sequences of two molecular clones of human herpesvirus 6 (HHV-6) (strain GS) and comparison with those of human cytomegalovirus (HCMV) has allowed the identification of the genes for the glycoprotein H (gH) and the putative large tegument protein of HHV-6. Two molecular clones of fragments of HHV-6, the BamHI-G fragment (7,981 bp) of the clone termed pZVB43 and a HindIII fragment (8,717 bp) of the clone termed pZVH14, represent approximately 10% of the HHV-6 genome (16,689). An open reading frame within the BamHI-G fragment was designated the gH gene of HHV-6 because of the extensive sequence similarity of its predicted product (79,549 Da) to the HCMV gH gene product. The predicted product (239,589 Da) of an open reading frame within clone pZVH14 showed homology to the predicted product of the proposed gene of HCMV representing the large tegument protein. Computer analyses indicated a closer relationship of the predicted peptides of these HHV-6 genes to those of HCMV than to those of the other human herpesviruses Epstein-Barr virus, herpes simplex virus type 1, and varicella-zoster virus. The gH gene was more conserved among the human herpesvirus group, while significant sequence similarity of the tegument gene could be found only with that of HCMV. The data reported here with one conserved gene (gH) and a more divergent gene (tegument) support previous reports that HHV-6 and HCMV are more closely related to each other than to the other well-characterized human herpesviruses.

Complete nucleotide sequences of functional clones of the AIDS virus.

To examine the mechanism of lymphocytotoxicity induced by human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), an in vitro model has been developed. Introduction of an HTLV-III/LAV proviral clone, HXB2, into normal lymphocytes results in the production of virions and cell death. The complete nucleotide sequence of the proviral form of HXB2 has now been determined. Its structure is quite similar to that previously determined for HTLV-III/LAV clones whose biological capacities had not previously been demonstrated. The biological function of two additional clones of HTLV-III/LAV, BH10 and HXB3, are reported. Clone BH10 which lacks the 5' long terminal repeat sequences (LTR) and a portion of the 3' LTR is reconstituted by substituting the corresponding sequences of HXB2 and is shown to be capable of generating infectious cytopathic virions. Clone HXB3, which has been partially sequenced, is also found to be capable of producing lymphocytopathic virus. Clone HXB3 differs from HXB2 in its lack of a termination codon in 3' orf, demonstrating that 3' orf plays no major role in virus replication or cytopathic activity. These data provide the necessary background to allow the identification of viral determinants of replication, cytopathic activity, and antigenicity using these functional proviral clones.

Activation of a novel KpnI transcript by downstream integration of a human T-lymphotropic virus type I provirus.

A cDNA library was constructed from the HUT102 cell line established from a patient with adult T-cell leukemia/lymphoma and screened for cDNA clones that contain (i) cellular sequences abundantly expressed in HUT102 cells and not in the virus-negative T-cell line HUT78, and (ii) viral long terminal repeat (LTR) sequences either in the 5' end or in the 3' end. One such cDNA clone, KT1, was isolated and its nucleotide sequence was determined. It contains three regions: a KpnI repeat, a unique cellular region (UCR), and the U3 + R sequence of the human T-lymphotropic virus type I LTR. The arrangement of this clone suggests that its RNA transcript was activated by provirus integration in cis, possibly by the activity of a downstream provirus enhancer. Analysis of HUT102 DNA shows that one allele of the KT1 UCR is rearranged. The expression of the KT1 UCR is unique to HUT102. These data are consistent with the idea that the human T-lymphotropic virus type I LTR contains an enhancer which can activate upstream sequences in cis. The possible significance of this finding is discussed.

Fine structure and evolution of the rat serum albumin gene.

The exons, their boundaries, and approximately half of the intronic deoxyribonucleic acid of the rat serum albumin gene were sequenced. In addition to the 14 exons identified earlier by R-loop analysis, a small exon was detected between the "leader" exon (Z) and exon B. The leader exon encoded the 5'-untranslated portion of albumin messenger ribonucleic acid and the "pre-pro" oligopeptide present on the nascent protein. The sites of initiation and termination of transcription were tentatively identified by comparison of the 5' and 3' gene-flanking sequences with those of other eucaryotic genes. All 28 intron/exon junctions conformed to the "GT-AG rule" (Breathnach et al., Proc. Natl. Acad. Sci. 75:4853-4857, 1978). The three homologous domains of albumin were encoded by three subgenes that consisted of four exons each and evolved by intragenic duplication of a common ancestor. The second and forth exons of each subgene appeared to be the result of an even earlier duplication event. We propose a model for the evolution of this gene that accounts for the observed patterns of exon size and homology.

Sequence homology between RNAs encoding rat alpha-fetoprotein and rat serum albumin.

We have determined the sequences of the recombinant DNA inserts of three bacterial plasmid cDNA clones containing most of the rat alpha a-fetoprotein mRNA. The resultant nucleotide sequence of alpha-fetoprotein was exhaustively compared to the nucleotide sequence of the mRNA encoding rat serum albumin. These two mRNAs have extensive homology (50%) throughout and the same intron locations. The amino acid sequence of rat alpha-fetoprotein has been deduced from the nucleotide sequence, and its comparison to rat serum albumin's amino acid sequence reveals a 34% homology. The regularly spaced positions of the cysteines found in serum albumin are conserved in rat alpha-fetoprotein, indicating that these two proteins may have a similar secondary folding structure. These homologies indicate that alpha-fetoprotein and serum albumin were derived by duplication of a common ancestral gene and constitute a gene family.

DNA-binding nonhistone proteins: DNA site reassociation.

The DNA-binding nonhistone proteins (NHP) have been demonstrated to fractionate the rat genome into protein-bound and unbound DNA sequences. Twenty percent of highly sheared rat DNA [approximately 350 base pair (bp)] can be retained on membrane filters as protein complexes. When extracted from the filter and retitrated with the NHP, a 4- to 5-fold enrichment of binding sites is present in the bound DNA with few, if any, sites detected in the unbound DNA. Rat DNA restricted by EcoRI endonuclease can be fractionated by its DNA-binding NHP retention characteristics. Reassociation kinetics of the bound restricted sequences indicate that 45.6% is a subset of total single-copy sequence of the rat genome an 26.9% is repetitive sequences. Cross hybridization studies indicate the repetitive sequences of the bound DNA are not enriched as much as the slow component of the rat genome. Thus a 4-fold enrichment of a subset of the rat genome has been observed via NHP-DNA interactions.