Chikungunya Virus' High Genomic Plasticity Enables Rapid Adaptation to Restrictive A549 Cells.

Chikungunya virus (CHIKV) is an emerging arthropod-borne virus that has spread globally during the last two decades. The virus is mainly transmitted by Aedes aegypti and Aedes albopictus mosquitos and is thus capable of replicating in both human and mosquito cells. CHIKV has a broad tropism in vivo, capable of replicating in various tissues and cell types but largely excluding blood cells. This was reflected in vitro by a broad array of adherent cell lines supporting CHIKV infection. One marked exception to this general rule is the resistance of the lung cancer-derived A549 cell line to CHIKV infection. We verified that A549 cells were restrictive to infection by multiple alphaviruses while being completely permissive to flavivirus infection. The adaptive growth of a primary CHIKV strain through multiple passages allowed the emergence of a CHIKV strain that productively infected A549 cells while causing overt cytopathic effects and without a fitness cost for replication in otherwise CHIKV-susceptible cells. Whole genome sequencing of polyclonal and monoclonal preparations of the adapted virus showed that a limited number of mutations consistently emerged in both structural (2 mutations in E2) and non-structural proteins (1 mutation in nsP1 and 1 mutation in nsP2). The introduction of the adaptive mutations, individually or in combinations, into a wild-type molecular clone of CHIKV allowed us to determine the relative contributions of the mutations to the new phenotype. We found that the mutations in the E2 envelope protein and non-structural proteins contributed significantly to the acquired phenotype. The nsP mutations were introduced in a split-genome trans-replicase assay to monitor their effect on viral genome replication efficiency. Interestingly, neither mutation supported increased viral genomic replication in either Vero or A549 cells.

A Chikungunya Virus trans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells.

Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV trans-replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects on trans-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced trans-replicase activity in mammalian cells, while in mosquito cells most of them increased trans-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both the trans-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus trans-replicase systems for studies of viral RNA replication and virus-host interactions.IMPORTANCE Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKV trans-replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKV trans-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

OBJECTIVES: The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. METHODS: Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. RESULTS: We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. CONCLUSIONS: These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

OBJECTIVES: Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. METHODS: From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. RESULTS AND CONCLUSIONS: We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

MiniCD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency.

BACKGROUND: Binding of the viral envelope protein (Env), and particularly of its gp120 subunit, to the cellular CD4 receptor is the first essential step of the HIV-1 entry process. The CD4 binding site (CD4bs) of gp120, and especially a recessed cavity occupied by the CD4 Phe43 residue, are known to be highly conserved among the different circulating subtypes and therefore constitute particularly interesting targets for vaccine and drug design. The miniCD4 proteins are a promising class of CD4bs inhibitors. Studying virus evolution under pressure of CD4bs inhibitors could provide insight on the gp120-CD4 interaction and viral entry. RESULTS: The present study reports on the resistance induction of two subtype B HIV-1 against the most active miniCD4, M48U1, and its ancestor, M48, and how these mutated positions affect CD4bs recognition, entry efficiency, and sensitivity to other CD4bs inhibitors. Resistance against M48U1 was always associated with S375R/N substitution in both BaL and SF162; M48 resistance was associated with D474N substitution in SF162 and with H105Y substitution in BaL. In addition, some other mutations at position V255 and G471 were of importance for SF162 resistant viruses. Except for 474, all of these mutated positions are conserved, and introducing them into an SF162 Env expressing infectious molecular clone (pBRNL4.3 SF162) resulted in decreased entry efficiency. Furthermore, resistant mutants showed at least some cross-resistance towards other CD4bs inhibitors, the V3 monoclonal antibody 447-52D and some even against the monoclonal antibody 17b, of which the epitope overlaps the co-receptor binding site. CONCLUSIONS: The mutations H105Y, V255M, S375R/N, G471R/E, and D474N are found to be involved in resistance towards M48 and M48U1. All mutated positions are part of, or in close proximity to, the CD4bs; most are highly conserved, and all have an impact on the entry efficiency, suggesting their importance for optimal virus infectivity.

HIV type 1 subtype A envelope genetic evolution in a slow progressing individual with consistent broadly neutralizing antibodies.

Studies of viruses taken from individuals with broad cross-neutralizing antibodies against primary isolates may reveal novel antibody specificities and their associated epitopes that could be useful for immunogen design. We report on the Env antigenic variability of a slow progressing HIV-1 subtype A-infected donor with consistent broad cross-neutralizing antibodies during the second decade of disease progression after vertical transmission. The Env evolution is characterized by a genetic shift to variants with altered V1-V5 loop sequences, marked by consecutive changes in V1, V4-V5, and C3 and largely conserved V2 and V3 loop sequences. Major V1 Env sequence expansion, variation by a duplication event, and cumulative addition of cysteine residues and potential N-glycosylation sites over time may contribute to escape from antibody pressure directed to Env receptor domains by changing the exposure of neutralization-sensitive epitopes. Conservation of functional epitopes may correlate with the continued presence of broad cross-neutralizing antibodies.

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.

Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins.

Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigen-loaded dendritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. In the present study, monocyte-derived DCs from nontreated HIV-1-seropositive patients were electroporated with codon-optimized ("humanized") mRNA encoding consensus HxB-2 (hHXB-2) Gag protein. These DCs elicited a strong HIV-1 Gag-specific interferon-gamma (IFN-gamma) response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DCs also triggered IFN-gamma secretion by autologous peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD8+ T cells from all patients tested. Next, a novel strategy was developed using autologous virus sequences. Significant specific IFN-gamma T-cell responses were induced in all patients tested by DCs electroporated with patients' autologous polymerase chain reaction (PCR)-amplified and in vitro-transcribed proviral and plasma viral mRNA encoding either Gag or Env. The stimulatory effect was seen on PBMCs, CD8+ T cells, and CD4+ T cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin-2 (IL-2) T-cell response was induced by DCs electroporated with hHxB-2 or proviral gag mRNA. These findings open a major perspective for the development of patient-specific immunotherapy for HIV-1 disease.