Devi Rajan

Semen-Derived Amyloid Fibrils Drastically Enhance HIV Infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.

Vpu serine 52 dependent counteraction of tetherin is required for HIV-1 replication in macrophages, but not in ex vivo human lymphoid tissue

BackgroundThe human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).ResultsWe show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.ConclusionOur data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.

Nef-Mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication Are Fundamental Properties of Primate Lentiviruses

ABSTRACT Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.

Human Immunodeficiency Virus Type 1 Variants Resistant to First- and Second-Version Fusion Inhibitors and Cytopathic in Ex Vivo Human Lymphoid Tissue

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) fusion inhibitors blocking viral entry by binding the gp41 heptad repeat 1 (HR1) region offer great promise for antiretroviral therapy, and the first of these inhibitors, T20 (Fuzeon; enfuvirtide), is successfully used in the clinic. It has been reported previously that changes in the 3-amino-acid GIV motif at positions 36 to 38 of gp41 HR1 mediate resistance to T20 but usually not to second-version fusion inhibitors, such as T1249, which target an overlapping but distinct region in HR1 including a conserved hydrophobic pocket (HP). Based on the common lack of cross-resistance and the difficulty of selecting T1249-resistant HIV-1 variants, it has been suggested that the determinants of resistance to first- and second-version fusion inhibitors may be different. To further assess HIV-1 resistance to fusion inhibitors and to analyze where changes in HR1 are tolerated, we randomized 16 codons in the HR1 region, including those making contact with HR2 codons and/or encoding residues in the GIV motif and the HP. We found that changes only at positions 37I, 38V, and 40Q near the N terminus of HR1 were tolerated. The propagation of randomly gp41-mutated HIV-1 variants in the presence of T1249 allowed the effective selection of highly resistant forms, all containing changes in the IV residues. Overall, the extent of T1249 resistance was inversely correlated to viral fitness and cytopathicity. Notably, one HIV-1 mutant showing ∼10-fold-reduced susceptibility to T1249 inhibition replicated with wild type-like kinetics and caused substantial CD4+-T-cell depletion in ex vivo-infected human lymphoid tissue in the presence and absence of an inhibitor. Taken together, our results show that the GIV motif also plays a key role in resistance to second-version fusion inhibitors and suggest that some resistant HIV-1 variants may be pathogenic in vivo.

Association of Nef with p21-Activated Kinase 2 Is Dispensable for Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity in Ex Vivo-Infected Human Lymphoid Tissue

ABSTRACT Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4+ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.

Effect of R77Q, R77A and R80A changes in Vpr on HIV-1 replication and CD4 T cell depletion in human lymphoid tissue ex vivo.

Background:It has been suggested that mutations of R77A and R80A in the HIV-1 viral protein R (Vpr) impair its proapoptotic activity and that a naturally occurring R77Q variation is associated with non-progressive HIV-1 infection. Rationale:To assess the effect of Vpr R77Q, R77A and R80A mutations on the efficiency of CCR5(R5)- and CXCR4(X4)-tropic HIV-1 replication and cytopathicity in human lymphoid tissue (HLT). Methods:Vpr mutants of the X4-tropic HIV-1 NL4-3 clone and an R5-tropic derivative were generated by PCR mutagenesis. Virus stocks established by transfection of 293T cells were used to infect macrophages and ex vivo HLT. HIV-1 replication was assessed by measuring p24 core antigen in the culture supernatants and CD4 T-cell depletion and apoptosis were measured by flow cytometric analysis. Results:The R5-tropic HIV-1 Vpr mutants replicated with slightly (R77A, R77Q) to moderately (R80A) reduced efficiency in ex vivo-infected HLT and macrophages. In comparison, the changes in Vpr had negligible effects on replication of the X4-tropic forms in lymphatic tissues. Mutation of R77Q and R80A reduced apoptosis of HIV-1-infected cells in ex vivo-infected HLT independently of the viral coreceptor tropism. However, only the R5-tropic HIV-1 Vpr mutants caused markedly less CD4 T-cell depletion than wild-type HIV-1 at the end of ex vivo HLT culture. Conclusions:The observation that Vpr R77Q reduces the cytopathicity of R5-tropic HIV-1 in lymphoid tissues supports a role in non-progressive HIV-1 infection but the attenuating effects might be dependent on the viral subtype and coreceptor tropism.

Response to Rhinovirus Infection by Human Airway Epithelial Cells and Peripheral Blood Mononuclear Cells in an In Vitro Two-Chamber Tissue Culture System

Human rhinovirus (HRV) infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells) and subsequent exposure of human peripheral blood mononuclear cells (PBMCs) to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B) and HRV 16 (species A) induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate novel approaches to its treatment.

Hsp70 Interacts with the Retroviral Restriction Factor TRIM5α and Assists the Folding of TRIM5α

Tripartite motif (TRIM) protein TRIM5α has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5α is not known. In this study, we showed that transiently overexpressed TRIM5α causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5α suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5α-binding partner. The interaction between Hsp70 and TRIM5α was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5α-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5α, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5α-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5α. Studies using a series of TRIM5α deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5α structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5α, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5α in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5α-induced cellular insults.

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.

Infant Viral Respiratory Infection Nasal-Immune-Response Patterns and Their Association with Subsequent Childhood Recurrent Wheeze

Rationale: Recurrent wheeze and asthma are thought to result from alterations in early life immune development following respiratory syncytial virus (RSV) infection. However, prior studies of the nasal immune response to infection have assessed only individual cytokines, which does not capture the whole spectrum of response to infection. Objectives: To identify nasal immune phenotypes in response to RSV infection and their association with recurrent wheeze. Methods: A birth cohort of term healthy infants born June to December were recruited and followed to capture the first infant RSV infection. Nasal wash samples were collected during acute respiratory infection, viruses were identified by RT‐PCR, and immune‐response analytes were assayed using a multianalyte bead‐based panel. Immune‐response clusters were identified using machine learning, and association with recurrent wheeze at age 1 and 2 years was assessed using logistic regression. Measurements and Main Results: We identified two novel and distinct immune‐response clusters to RSV and human rhinovirus. In RSV‐infected infants, a nasal immune‐response cluster characterized by lower non‐IFN antiviral immune‐response mediators, and higher type‐2 and type‐17 cytokines was significantly associated with first and second year recurrent wheeze. In comparison, we did not observe this in infants with human rhinovirus acute respiratory infection. Based on network analysis, type‐2 and type‐17 cytokines were central to the immune response to RSV, whereas growth factors and chemokines were central to the immune response to human rhinovirus. Conclusions: Distinct immune‐response clusters during infant RSV infection and their association with risk of recurrent wheeze provide insights into the risk factors for and mechanisms of asthma development.