The effects of MAPK p38α on AZT resistance against reactivating HIV-1 replication in ACH2 cells.

Antiretroviral therapy (ART) has remarkably decreased HIV-related mortality. However, drug-resistant HIV variants pose a potential threat to the long-term success of ART. Both HIV mutants and host factors can cause HIV drug resistance. Using susceptible ACH2 cells chronically infected with HIV-1, we examined the effects of MAPK p38α on AZT resistance against reactivating HIV-1 replication that can be activated by HIV-1 superinfection. We found that HIV-1 superinfection induced more viral production, which was diminished by p38 inhibitor, SB203580, and by AZT in cells infected with non-AZT-resistant HIV-1 strain MN. p38α expression can resist action of AZT in inhibition of HIV-1 replication with increased expression of transcription factor, NF-ĸBp65, SP1, and c-Fos through activation of TCR-related pathways with upregulation of CD3, TCRα, TCRß, Zap-70, PKC, PLCγ1, GRB2, and PI3K/Akt expression. In HIV-1 MN superinfection under AZT treatment, expression of p38α led to HIV vif expression and inhibited APOBEC3G expression. We also investigated effects of p38α on gp130/JAK-STAT pathways, in which p38α increased expression of protein, gp130, EGFR, Jak2, STAT1, STAT3, STAT5, ras, and TF. p38α could induce apoptotic pathways with upregulation of Fas, FADD, Caspase-8, p53, and Bax, and downregulation of Bcl2 expression. These results indicate that p38α plays a positive role in reactivation of viral replication from HIV-1 latent infection and leads to HIV-1 AZT resistance. In conclusion, MAPKp38α can activate HIV-1 replication inhibited by AZT from HIV-1 latent infection and may be used as a latency reversal agent. The activation involves induction of several cell signaling pathways that are required for HIV-1 replication, which may be integrated into future viral remission strategies.

Differences in Activation of HIV-1 Replication by Superinfection With HIV-1 and HIV-2 in U1 Cells.

Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV-1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV-1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV-1 and HIV-2 in macrophages. We found that HIV-1 (MN) superinfection displayed increased HIV-1 replication in a time-dependent manner; while cells infected with HIV-2 (Rod) initially showed increased HIV-1 replication, followed by a decrease in HIV-1 RNA production. HIV-1 superinfection upregulated/activated NF-ĸB, NFAT, AP-1, SP-1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRß, Zap-70, PLCγ1, and PKCΘ in T cell receptor-related signaling pathways; while HIV-2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated acetyl-histone H3 and histone H3 (K4), while HIV-2 superinfection demonstrated an increase in HDAC1 and a decrease in acetyl-histone H3 and histone H3 (K4) relative to HIV-1 superinfection. U1 cells superinfected with HIV-1 or HIV-2 showed differential expression of proteins, IL-2, PARP-1, YB-1, and LysRS. These findings indicate that superinfection with HIV-1 or HIV-2 has different effects on reactivation of HIV-1 replication. HIV-1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV-2 superinfection. Cells infected with HIV-2 showed lower level of HIV-1 replication, suggesting that co-infection with HIV-2 may result in slower progression toward AIDS. J. Cell. Physiol. 232: 1746-1753, 2017. © 2016 Wiley Periodicals, Inc.

IL-1ß and IL-18 inhibition of HIV-1 replication in Jurkat cells and PBMCs.

HIV-1 infection-induced apoptosis is able to ensure viral replication. The death of some CD4+ T cells residing in lymphoid tissues can be induced by HIV-1 infection through caspase-1 driven pyroptosis with release of cytokine of IL-1ß and IL-18. It is not well known whether IL-1ß and IL-18 affect HIV-1 replication in lymphocytic cells. Using susceptible lymphocytic cell line, Jurkat cells, and primary peripheral blood mononuclear cells (PBMCs), we studied the effects of IL-1ß and IL-18 on HIV-1 replication. We found that treatment with exogenous IL-1ß protein (rIL-1ß) and IL-18 protein (rIL-18), or expression of IL-1ß and IL-18 significantly reduced HIV-1 replication. HIV-1 infection enhanced caspase-3 expression and its activation, and had no effects on caspase-1 activity. Treatment with rIL-1ß and rIL-18 dramatically lowered caspase-3 activity. IL-1ß and IL-18 also played roles in diminishing reactivation of viral replication from latency in J1.1 cells. These results indicate that IL-1ß and IL-18 are able to inhibit HIV-1 replication, and their effects may be due to signaling through apoptosis involved in inactivation of caspase-3 activity.

Nanomicroarray and multiplex next-generation sequencing for simultaneous identification and characterization of influenza viruses.

Conventional methods for detection and discrimination of influenza viruses are time consuming and labor intensive. We developed a diagnostic platform for simultaneous identification and characterization of influenza viruses that uses a combination of nanomicroarray for screening and multiplex next-generation sequencing (NGS) assays for laboratory confirmation. The nanomicroarray was developed to target hemagglutinin, neuraminidase, and matrix genes to identify influenza A and B viruses. PCR amplicons synthesized by using an adapted universal primer for all 8 gene segments of 9 influenza A subtypes were detected in the nanomicroarray and confirmed by the NGS assays. This platform can simultaneously detect and differentiate multiple influenza A subtypes in a single sample. Use of these methods as part of a new diagnostic algorithm for detection and confirmation of influenza infections may provide ongoing public health benefits by assisting with future epidemiologic studies and improving preparedness for potential influenza pandemics.

A highly sensitive europium nanoparticle-based immunoassay for detection of influenza A/B virus antigen in clinical specimens.

We report the development of a novel europium nanoparticle-based immunoassay (ENIA) for rapid detection of influenza A and influenza B viruses. The ENIA demonstrated sensitivities of 90.7% (147/162) for influenza A viruses and 81.80% (9/11) for influenza B viruses compared to those for an in-house reverse transcription (RT)-PCR assay in testing of influenza-positive clinical samples.

Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs.

Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.

Highly pathogenic avian influenza A virus (H5N1) can be transmitted in ferrets by transfusion.

BACKGROUND: Highly pathogenic avian influenza A virus has been shown to infect organs other than the lung, and this is likely to be mediated by systemic spread resulting from viremia which has been detected in blood in severe cases of infection with avian H5N1 viruses. The infectivity of virus in blood and the potential for virus transmission by transfusion has not been investigated. METHODS: Using a susceptible ferret animal model, we evaluated viremia and transmission by blood transfusion. Blood was collected on day 2, 4, 6, and 10 post-infection (or before death) from donor ferrets infected with either low dose (1.0 × 10(2.6) EID50/ml) or high dose (1.0 × 10(3.6) EID50/ml) of H5N1 virus, A/VN/1203/04 and transfused to recipient animals. RESULTS: Viremia was observed in 2/12 (16.67%) recipients that received blood from donor ferrets infected with low dose and 7/12 (58.33%) recipients who received blood from high dose infected donors. 1/12 (8.3%) low dose recipients and 6/12 (50%) high dose recipients died within 11 days after transfusion. Increased changes in body weight and temperatures were observed in high dose recipients, and high levels of viral RNA were detected in recipient ferrets after transfusion of blood from the early viremic phase, which also correlated with adverse impact on their survival. CONCLUSION: These data suggest that highly pathogenic avian influenza A virus, H5N1, is transmissible by blood transfusion in ferrets. Low levels of viremia were detected around the time of onset of symptoms and later in ferrets infected with highly pathogenic H5N1 virus. These findings may have implication for pathogenesis and transmissibility of H5N1.

Some mechanisms of FLIP expression in inhibition of HIV-1 replication in Jurkat cells, CD4+ T cells and PBMCs.

HIV-1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV-1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV-1 replication are critical to understanding the mechanisms of HIV-1 replicative cycle. Using the susceptible Jurkat cell line, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase-8, in HIV-1 production. Full length cellular FLIP (cFLIP) inhibited HIV-1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV-1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV-1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas-mediated apoptotic pathway. The inactivated FADD also inhibited HIV-1 replication.

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells.

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells.

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X(L), and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.

Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected blood donors or individuals in Africa.

BACKGROUND: Since the identification of xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region. STUDY DESIGN AND METHODS: A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay. RESULTS: Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive. CONCLUSIONS: Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.

Identification of new, emerging HIV-1 unique recombinant forms and drug resistant viruses circulating in Cameroon.

BACKGROUND: The HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients. METHODOLOGY: Blood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database. RESULTS: Phylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies. CONCLUSIONS: Our results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.

Stability and infectivity of novel pandemic influenza A (H1N1) virus in blood-derived matrices under different storage conditions.

BACKGROUND: Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied. METHODS: We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID(50) assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C. RESULTS: Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low. CONCLUSION: These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples.

Identification, Genetic Characterization and Validation of Highly Diverse HIV-1 Viruses for Reference Panel Development.

The continued diversification of HIV poses potentially significant challenges to HIV diagnostics and therapeutics. The dynamic evolution of emerging variants is highlighted in countries such as Cameroon in West Central Africa, where all known subtypes and circulating recombinant forms (CRFs) have been shown to be prevalent. We obtained several hundred HIV-positive plasma and viruses from this region for characterization and identification of highly divergent HIV strains. A total of 163 viral strains were cultured to high titers and high volumes using donor peripheral blood mononuclear cells (PBMCs). Initially, 101 viruses representing 59 strains were well characterized and categorized. Results showed that the viral load (VL) range was 0.36-398.9 × 107 copies/mL, p24 values was 0.2-1134 ng/mL. Phylogenetic analysis of thirty-six near full-length HIV-1 genomic sequences demonstrated that most recombinants were highly diverse CRF02 containing unique recombinant forms (URFs). There were seven viral isolates identified as pure subtype/sub-subtypes (F2, A1, G, and D), six as CRFs (CRF06, CRF18, and CRF22), and ten as URFs. These extensively characterized reagents reflect the current dynamic and complex HIV epidemic in Cameroon and provide valuable insights into the potential phylogenetic evolutionary trend of global HIV molecular epidemiology in the future. These materials may be useful for development of HIV validation and reference panels to evaluate the performance of serologic antigen and nucleic acid assays for their ability to detect and quantitate highly divergent HIV strains.

Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay.

BACKGROUND: For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2). RESULTS: Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 103 TCID50 units for H5N1 viral RNA. CONCLUSIONS: The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.

HIV-1 reverse transcriptase drug-resistance mutations in chronically infected individuals receiving or naïve to HAART in Cameroon.

The most common first-line, highly active anti-retroviral therapy (HAART) received by individuals infected with HIV-1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non-B HIV-1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty-nine HIV-1-positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South-West and North-West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug-resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug-naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug-naïve individuals, 24% harbored viruses with drug-resistance mutations, suggesting that HIV-1 drug-resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first-line HAART, coupled with the high prevalence of HIV-1 variants with drug-resistance mutations among drug-naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second-line HAART strategy in Cameroon.

Changes in the level of apoptosis-related proteins in Jurkat cells infected with HIV-1 versus HIV-2.

Human immunodeficiency virus (HIV) infection-induced apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 T cells and other types of cells is a major factor in the pathogenesis of AIDS. Clinically, HIV-2 patients have a higher CD4 cell count at the time of an AIDS diagnosis, and generally have longer survival after development of symptoms. The mortality after an AIDS diagnosis has been reported to be more influenced by CD4 cell count than HIV type. Previous studies have shown significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes; however, the relative contributions of HIV-1 and HIV-2 infection leading to cell death remain unclear. Using a human cell line, Jurkat, we examined differences in key molecules involved in apoptotic signaling pathways during infection with either HIV-1 or HIV-2. HIV-1 infection generated more reactive oxygen species (ROS), increased the expression of a larger number of molecules involved in cell signaling such as p47, p38alpha, JNK, c-Yes, total PKC, and decreased the expression of molecules such as p38beta, ERK1/2, and XIAP relative to HIV-2 infection. HIV-1 induced a higher degree of cell death through stronger activation of both apoptotic pathways. HIV-1 infection downregulated both Bcl-X(L) and FLIP expressions at later time points postinfection, while HIV-2 infection dramatically upregulated both Bcl-X(L) and FLIP expression. We also found that the expression of Bcl-X(L) or FLIP resulted in significant inhibition of HIV replication in Jurkat cells. These findings suggest that HIV-1 infection with high levels of cytotoxicity results in a higher level of cell death through apoptosis during a short time postinfection. The longer period of infection observed with HIV-2 with a lower degree of cytotoxicity was accompanied by increased Bcl-X(L) and FLIP expression. High protein levels of Bcl-X(L) or FLIP inhibit HIV replication and may be one explanation for the clinical observation that HIV-2 infected patients generally tend to be long-term nonprogressors with high CD4 lymphocyte counts compared with HIV-1 infected persons.

Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms.

The critical role of the regulatory elements at the 5' end of the HIV-1 genome in controlling the life cycle of HIV-1 indicates that this region significantly influences virus fitness and its biological properties. In this study, we performed a detailed characterization of strain-specific variability of sequences from the U5 to upstream of the gag gene start codon of diverse HIV-1 strains by using next-generation sequencing (NGS) techniques. Overall, we found that this region of the HIV-1 genome displayed a low degree of intra-strain variability. On the other hand, inter-strain variability was found to be as high as that reported for gag and env genes (13-17%). We observed strain-specific single point and clustered mutations in the U5, PBS, and gag leader sequences (GLS), generating potential strain-specific transcription factor binding sites (TFBS). Using an infrared gel shift assay, we demonstrated the presence of potential TFBS such as E-box in CRF22_01A, and Stat 6 in subtypes A and G, as well as in their related CRFs. The strain-specific variation found in the sequence corresponding at the RNA level to functional domains of the 5' UTR, could also potentially impact the secondary/tertiary structural rearrangement of this region. Thus, the variability observed in this 5' end of the genomic region of divergent HIV-1 strains strongly suggests that functions of this region might be affected in a strain-specific manner. Our findings provide new insights into DNA-protein interactions that regulate HIV-1 replication and the influence of strain characterization on the biology of HIV-1 infection.

c-FLIPL regulates PKC via AP-2 to inhibit Bax-mediated apoptosis induced by HIV-1 gp120 in Jurkat cells.

c-FLIPL, an inhibitor of caspase 8, is known to inhibit the Fas/caspase 8 apoptotic pathway; however, its involvement of Bax/mitochondrial apoptosis is not well understood. Using human cells, Jurkat cell line, induced with HIV-1 gp120, we studied the effects of c-FLIPL on Bax/mitochondrial apoptosis. We found that the induction of apoptosis by HIV-1 envelope protein, gp120, involved the activation of both Bax-dependent and death receptor-mediated pathways, and HIV-1 infection deceased c-FLIPL expression. Interestingly, c-FLIPL expression downregulated protein kinase C (PKC) expression at the transcript level involving activated protein-2 (AP-2). c-FLIPL expression reduced AP-2 protein levels required to promote PKC protein expression and PKC-associated inactive form of Bax, and inhibited Bax activation, suggesting that c-FLIPL inhibits Bax activation via modulating PKC expression at the transcriptional level involving AP-2 during gp120 treatment. Collectively, these findings further corroborate the concept that gp120 plays an important role, via involvement of molecules such as c-FLIPL, in apoptotic cell death due to HIV-1 infection.

Comparative analysis of cell culture and prediction algorithms for phenotyping of genetically diverse HIV-1 strains from Cameroon.

BACKGROUND: With the advent of entry inhibitors, monitoring of viral tropism in the clinical setting is important. Conventional methods are cell-based and lengthy, therefore V3 sequence based prediction algorithms are becoming increasingly attractive as monitoring tools. Here we report a comparative analysis of viral tropism of strains circulating in Cameroon where diverse and emerging variant strains are prevalent. METHODS: Viruses were isolated from 17 HIV positive individuals from three cities in Cameroon. Ghost cell lines expressing either CCR5 or CXCR4 with CD4 or CD4 alone (NIH AIDS Reagent Program) were used to determine co-receptor usage. HIV replication was determined by measuring p24 antigen levels. Plasma viral load (VL) was determined using the Versant bDNA assay. Nucleotide sequencing was performed on the V3 region and sequences were edited, aligned and translated into amino acids as described in the algorithm. Bio-informatics tools based on the 11/25 and charge rule were used to predict co-receptor usage. RESULTS: The majority of patient isolates in our study were CRF02_AG or CRF02_AG containing recombinants. Tropism of these complex viruses based on the cell culture assay was determined to be R5 in 15/17 (88.2%) patients. However, two patient isolates were dual tropic R5X4 and had drug-specific mutations. Of these two patients, one was on antiretroviral treatment with a VL of 20,899 copies/ml and the other was drug-naïve with 141,198 copies/ml. Genotype based prediction was overall in good agreement with phenotype for R5 viruses, where 93% (14/15) of results were comparable, dual tropic viruses being reported as X4 viruses by prediction. CONCLUSION: Our results indicate that most HIV strains in Cameroon were R5 tropic and some harbored drug-resistant mutations. V3 sequence based prediction compared well with cell based assays for R5 strains and may be useful even in settings where highly diverse strains are prevalent.