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  • 1
    Online Resource
    Online Resource
    Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma
    American Society for Microbiology ; 2019
    In:  Journal of Virology Vol. 93, No. 1 ( 2019-01)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 93, No. 1 ( 2019-01)
    Abstract: Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requires a priori sequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i) de novo genome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory gene vpx in all patients. Our study demonstrates that RNA-Seq is a feasible full-genome de novo sequencing method for blood plasma samples collected from HIV-2-infected individuals. IMPORTANCE An accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detailed a priori sequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.00677-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 1495529-5
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  • 2
    Online Resource
    Online Resource
    Lessons from the Genome Sequence of Neurospora crassa : Tracing the Path from Genomic Blueprint to Multicellular Organism
    American Society for Microbiology ; 2004
    In:  Microbiology and Molecular Biology Reviews Vol. 68, No. 1 ( 2004-03), p. 1-108
    Details
    In: Microbiology and Molecular Biology Reviews, American Society for Microbiology, Vol. 68, No. 1 ( 2004-03), p. 1-108
    Abstract: We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa . Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.
    Type of Medium: Online Resource
    ISSN: 1092-2172 , 1098-5557
    URL: Article
    DOI: 10.1128/MMBR.68.1.1-108.2004
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 2026768-X
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Collaboration of a Detrimental HLA-B*35:01 Allele with HLA-A*24:02 in Coevolution of HIV-1 with T Cells Leading to Poorer Clinical Outcomes
    American Society for Microbiology ; 2021
    In:  Journal of Virology Vol. 95, No. 23 ( 2021-11-09)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 95, No. 23 ( 2021-11-09)
    Abstract: Although mutant-specific T cells are elicited in some individuals infected with HIV-1 mutant viruses, the detailed characteristics of these T cells remain unknown. A recent study showed that the accumulation of strains expressing Nef135F, which were selected by HLA-A*24:02-restricted T cells, was associated with poor outcomes in individuals with the detrimental HLA-B*35:01 allele and that HLA-B*35:01-restricted NefYF9 (Nef135-143)-specific T cells failed to recognize target cells infected with Nef135F mutant viruses. Here, we investigated HLA-B*35:01-restricted T cells specific for the NefFF9 epitope incorporating the Nef135F mutation. Longitudinal T-cell receptor (TCR) clonotype analysis demonstrated that 3 types of HLA-B*35:01-restricted T cells (wild-type [WT] specific, mutant specific, and cross-reactive) with different T cell repertoires were elicited during the clinical course. HLA-B*35:01 + individuals possessing wild-type-specific T cells had a significantly lower plasma viral load (pVL) than those with mutant-specific and/or cross-reactive T cells, even though the latter T cells effectively recognized the mutant virus-infected cells. These results suggest that mutant-specific and cross-reactive T cells could only partially suppress HIV-1 replication in vivo . An e x vivo analysis of the T cells showed higher expression of PD-1 on cross-reactive T cells and lower expression of CD160/2B4 on the mutant-specific T cells than other T cells, implying that these inhibitory and stimulatory molecules are key to the reduced function of these T cells. In the present study, we demonstrate that mutant-specific and cross-reactive T cells do not contribute to the suppression of HIV-1 replication in HIV-1-infected individuals, even though they have the capacity to recognize mutant virus-infected cells. Thus, the collaboration of HLA-A*24:02 with the detrimental allele HLA-B*35:01 resulted in the coevolution of HIV-1 alongside virus-specific T cells, leading to poorer clinical outcomes. IMPORTANCE HIV-1 escape mutations are selected under pressure from HIV-1-specific CD8 + T cells. Accumulation of these mutations in circulating viruses impairs the control of HIV-1 by HIV-1-specific T cells. Although it is known that HIV-1-specific T cells recognizing mutant virus were elicited in some individuals infected with a mutant virus, the role of these T cells remains unclear. Accumulation of phenylalanine at HIV-1 Nef135 (Nef135F), which is selected by HLA-A*24:02-restricted T cells, led to poor clinical outcome in individuals carrying the detrimental HLA-B*35:01 allele. In the present study, we found that HLA-B*35:01-restricted mutant-specific and cross-reactive T cells were elicited in HLA-B*35:01 + individuals infected with the Nef135F mutant virus. These T cells could not effectively suppress HIV-1 replication in vivo even though they could recognize mutant virus-infected cells in vitro . Mutant-specific and cross-reactive T cells expressed lower levels of stimulatory molecules and higher levels of inhibitory molecules, respectively, suggesting a potential mechanism whereby these T cells fail to suppress HIV-1 replication in HIV-1-infected individuals.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.01259-21
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 1495529-5
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