Dendritic cell-lymphocyte cross talk downregulates host restriction factor SAMHD1 and stimulates HIV-1 replication in dendritic cells.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-α]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture. IMPORTANCE: SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DCs and lymphocytes mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with downregulation of SAMHD1 expression and activation of innate immune sensing by DCs. We propose that DC-lymphocyte cross talk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing.

Neutralizing antibodies inhibit HIV-1 transfer from primary dendritic cells to autologous CD4 T lymphocytes.

Dendritic cells (DCs) support only low levels of HIV-1 replication, but have been shown to transfer infectious viral particles highly efficiently to neighboring permissive CD4 T lymphocytes. This mode of cell-to-cell HIV-1 spread may be a predominant mode of infection and dissemination. In the present study, we analyzed the kinetics of fusion, replication, and the ability of HIV-1-specific Abs to inhibit HIV-1 transfer from immature DCs to autologous CD4 T lymphocytes. We found that neutralizing mAbs prevented HIV-1 transfer to CD4 T lymphocytes in trans and in cis, whereas nonneutralizing Abs did not. Neutralizing Abs also significantly decreased HIV-1 replication in DCs, even when added 2 hours after HIV-1 infection. Interestingly, a similar inhibition of HIV-1 replication in DCs was detected with some nonneutralizing Abs and was correlated with DC maturation. We suggest that the binding of HIV-1-specific Abs to FcγRs leads to HIV-1 inhibition in DCs by triggering DC maturation. This efficient inhibition of HIV-1 transfer by Abs highlights the importance of inducing HIV-specific Abs by vaccination directly at the mucosal portal of HIV-1 entry to prevent early dissemination after sexual transmission.

Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene.

BACKGROUND: Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. RESULTS: The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. CONCLUSIONS: This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide.

Stimulation of HIV-1 replication in immature dendritic cells in contact with primary CD4 T or B lymphocytes.

Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence.

Multiplex PCR and quality control of Epinotia aporema granulovirus production.

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1-3 sets of selected 5-6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.