Deep gene sequence cluster analyses of multi-virus infected mucosal tissue reveal enhanced transmission of acute HIV-1

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multi-virus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue while Envacute viruses penetrated the human tissue and established infection of CD4(+) T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggests that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g. acute Env HIV-1) can escape this trapped replication for systemic infection. Importance: During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa and leading to systemic infection of a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance to understanding dynamics of infections and to now design focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early as compared to chronic infection can more efficiently traverse the mucosal epithelium and transmitted to T cells, suggesting higher transmission fitness. These studies focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap

Role of Gp120 Glycosylation in Sexual Transmission of HIV

Background: In chronic HIV patients, the viral populations are genetically diverse due to mutations introduced by the viral reverse transcriptase during HIV replication. However, more than 80% new infections result from single transmission founder (TF) viruses; therefore, targeting the TFs is key to control AIDS worldwide. Gp120 is a glycosylated envelope protein required for HIV infection, propagation, and transmission. Glycans on gp120 influence HIV infectivity through their interactions with lectins, the carbohydrate-binding immune proteins in the host mucosa. To transmit sexually, viruses must overcome the lectin traps to access more target T cells. Hypothesis: TF viruses are less likely to be trapped by host lectins due to their reduced gp120 glycosylation, thus more infectious. Methods: We aim to characterize and compare the gp120 glycosylation signatures in TF and chronic HIV strains, B4 and Q0 respectively, using mass spectrometry (MS), surface plasmon resonance (SPR), and capillary electrophoresis (CE). To date, we have established a work flow to purify gp120 glycoproteins, perform MS using ETHcD methods, and analyze raw MS data using the GlycoPAT software. We are currently analyzing MS data for three replicates of B4 and the first replicate of Q0. Then we will compare the glycosylation patterns between the two strains. CE and SPR will be performed to test the glycan enrichment and functional interactions between gp120 and lectins, respectively. Discussion: Our results will provide qualitative and quantitative details about gp120 glycosylation underlying the strong infectivity of TF viruses, shedding light on new strategies to develop HIV vaccines

Lifecycle driven planning of infrastructure:Public and private experiences with more integrated approaches for managing project complexity

Currently, many initiatives are under implementation in the Netherlands to integrate the stages of policymaking, plan development, construction, and operations and maintenance in the lifecycle of the infrastructure planning process by more explicitly involving business organizations. However, generally speaking, these integration initiatives stand alone and only connect a maximum of two stages at a time. In this article we explore whether and how contemporary lifecycle integration initiatives could be combined into a more integrated approach to be better able to address infrastructure planning complexities. We provide a framework for dealing with project complexity that distinguishes internal complexity, defined as the interrelatedness between project components, and external complexity, defined as the interaction of the project with its context. After assessing public and private experiences in combining single integration initiatives in complex settings by means of two focus group discussions, we conclude that current initiatives that connect stages in the planning process are suitable for addressing internal complexity. However, external complexity proves to be more difficult to adequately tackle when combining these lifecycle integration initiatives. We therefore recommend applying a more dynamic process management approach that stimulates continuous public-private interaction throughout the stages of the planning lifecycle. This could be facilitated by introducing alliances and cross-functional public-private teams

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3\u27 end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).<br /

Two-year follow-up of macaques developing intermittent control of the human immunodeficiency virus homolog simian immunodeficiency virus SIVmac251 in the chronic phase of infection

Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy and two experimental drugs, i.e., auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent posttherapy viral load control in chronically SIVmac251-infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4(+) T cells at viral rebound. Moreover, these macaques consistently displayed low-level simian immunodeficiency virus (SIV) diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in 2 years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir

Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

<p>Abstract</p> <p>Background</p> <p>Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.</p> <p>Results</p> <p>Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (<it>env</it>) gene as well as a siRNA specific for a downstream target sequence in the subtype D <it>env </it>gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.</p> <p>Conclusion</p> <p>These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.</p