Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach ...requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
HIV needs to be fit to transmitAlthough you might not think it, it's hard to catch HIV. Less than 1% of unprotected sexual exposures result in infection. What then leads to transmission? Carlson et ...al. determined the amino acid sequence of viruses infecting 137 Zambian heterosexual couples in which one partner infected the other (see the Perspective by Joseph and Swanstrom). The authors then used statistical modeling and found that transmitted viruses are typically the most evolutionarily fit. That is, compared to other viral variants in the infected person, the transmitted virus most closely matches the most common viral sequence found in the Zambian population.Science, this issue 10.1126/science.1254031; see also p. 136
Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral ...characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine ...lineage, and two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. To help identify determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 H1N1 (NL602), into the background of A/Puerto Rico/8/1934 H1N1 (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA, and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility.
Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.
HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the ...factors that influence HIV-1–induced immunopathology and subsequent CD4 ⁺ T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8 ⁺ T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4 ⁺ T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1–related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.
Significance HIV infection is associated with elevated inflammation and aberrant cellular immune activation. Indeed, the activation status of an HIV-infected individual is often more predictive of disease trajectory than viral load. Here, we highlight the importance of the replicative fitness of the transmitted viral variant in driving an early inflammatory state, characterized by T-cell activation and immune dysfunction. This impact on T-cell homeostasis is independent of protective host immune response genes and viral load. Highly replicating transmitted variants were also significantly more efficient at infecting memory CD4 ⁺ T cells, a population important for maintaining the latent viral reservoir. Together, these data provide a mechanism whereby viral replicative fitness acts as a major determinant of disease progression and persistence.
Alveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals ...living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.
The activation of naïve T cells requires antigen presentation by dendritic cells (DCs), and the process of antigen presentation is regulated over the course of DC maturation. One key aspect of this ...regulation is the cell surface up-regulation upon DC maturation of peptide·MHC-II complexes and the costimulatory molecule CD86. It is now clear that these critical induction events involve changes in ubiquitin-dependent trafficking of MHC-II and CD86 by the E3 ligase membrane-associated RING-CH-1 (MARCH1). Although ubiquitin-dependent trafficking of MHC-II has been well characterized, much less is known regarding the post-transcriptional regulation of CD86 expression. Here, we examined the physical and functional interaction between CD86 and MARCH1. We observed that CD86 is rapidly endocytosed in the presence of MARCH1 followed by lysosome-dependent degradation. Furthermore, we found that the association between CD86 and MARCH1 was conferred primarily by the transmembrane domains of the respective proteins. In contrast to MHC-II, which has a single, conserved ubiquitin acceptor site in the cytosolic domain, we found that multiple lysine residues in the cytosolic tail of CD86 could support ubiquitination consistent with the relative lack of sequence conservation across species within the CD86 cytosolic domain. These findings suggest that MARCH1 recruits multiple substrates via transmembrane domain-mediated interactions to permit substrate ubiquitination in the face of diverse cytosolic domain sequences.
Background: The ubiquitin ligase membrane-associated RING-CH-1 (MARCH1) regulates expression of key immune molecules.
Results: MARCH1 induces endocytosis and degradation of CD86 by ubiquitination of multiple lysine residues in CD86.
Conclusion: Functional MARCH1-CD86 interaction occurs despite poor sequence conservation in the relevant domains of CD86.
Significance: MARCH1 substrates are recruited via transmembrane domain-mediated interactions, permitting substrate ubiquitination despite poor conservation in cytosolic domains.
The genetic diversity of HIV impedes vaccine development. Identifying the viral properties of transmitted/founder (T/F) variants may provide a common vaccine target. To study the biological nature of ...T/F viruses, we constructed full-length clones from women detected during Fiebig stage I acute HIV-1 infection (AHI) from heterosexual male-to-female (MTF) transmission; and clones after one year of infection using In-Fusion-based cloning. Eighteen full-length T/F clones were generated from 9 women and six chronic infection clones were from 2 individuals. All clones but one were non-recombinant subtype C. Three of the 5 T/F clones and 3 chronic clones tested replicated efficiently in PBMCs and utilised CCR5 coreceptor for cell entry. Transmitted/founder and chronic infection clones displayed heterogenous in vitro replicative capacity and resistance to type I interferon. T/F viruses had shorter Env glycoproteins and fewer N-linked glycosylation sites in Env. Our findings suggest MTF transmission may select viruses with compact envelopes.
•We generated infectious molecular clones of HIV-1 subtype C transmitted/founder (T/F) and chronic infection variants.•The HIV-1 subtype C infectious clones display heterogeneity in in vitro replicative capacity and resistance to type I interferon.•T/F clones predominantly use the CCR5 coreceptor; shorter Env variable regions and fewer glycans compared to chronic viruses.
Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error ...rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.
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