The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.DOI: http://dx.doi.org/10.7554/eLife.15528.001

We have recently reported the isolation of a novel virus, provisionally designated C/swine/Oklahoma/1334/2011 (C/OK), with 50% overall homology to human influenza C viruses (ICV), from a pig in Oklahoma. Deep RNA sequencing of C/OK virus found a matrix 1 (M1) protein expression strategy that differed from that of ICV. The novelty of C/OK virus prompted us to investigate whether C/OK virus could exist in a nonswine species. Significantly, we found that C/OK virus was widespread in U.S. bovine herds, as demonstrated by reverse transcription (RT)-PCR and serological assays. Genome sequencing of three bovine viruses isolated from two herds in different states further confirmed these findings. To determine whether swine/bovine C/OK viruses can undergo reassortment with human ICV, and to clarify the taxonomic status of C/OK, in vitro reassortment and serological typing by agar gel immunodiffusion (AGID) were conducted. In vitro reassortment using two human ICV and two swine and bovine C/OK viruses demonstrated that human ICV and C/OK viruses were unable to reassort and produce viable progeny. Antigenically, no cross-recognition of detergent split virions was observed in AGID between human and nonhuman viruses by using polyclonal antibodies that were reactive to cognate antigens. Taken together, these results demonstrate that C/OK virus is genetically and antigenically distinct from ICV. The classification of the new virus in a separate genus of the Orthomyxoviridae family is proposed. The finding of C/OK virus in swine and bovine indicates that this new virus may spread and establish infection in other mammals, including humans.

The internal -methyladenosine (mA) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to mA-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the mA modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the mA-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered mA modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the mA modification of HIV-1 RNA affects viral replication.

The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.Transcription and replication of the influenza A viral genome involves a complex set of enzymatic reactions catalyzed by a heterotrimeric complex that is composed of three subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) (7,25,37). The PB1 subunit plays a central role in both polymerase and endonuclease activity (2, 26), the PB2 subunit has cap-binding activity and is responsible for the initiation of transcription (6,7,11), and the PA subunit has been suggested to function in both transcription and replication (4,5,30,33), but its exact role remains undetermined.In order for the individual polymerase proteins to perform their various functions, the three subunits need to come together to form a viral RNA polymerase complex; however, a significant proportion of polymerase dimers and oligomers can be detected in vitro (23). Our knowledge of the viral RNA polymerase complex assembly has increased over the past several years primarily by characterizing protein-protein interactions among the individual polymerase subunits (4,5,10,30,36,38,39,45,48). It is believed that the PB1 subunit forms the core of the polymerase complex. PB1 utilizes its N-terminal region to interact with PA, while PB1 binds PB2 through its C-terminal region. A specific interaction between PA and PB2 subunits has yet to be detected (36).Two models have been proposed to explain the assembly pathway and nuclear import of the polymerase complex (PA-PB1-PB2) (4,...

Despite significant advances in antiretroviral therapy, increasing drug resistance and toxicities observed among many of the current approved human immunodeficiency virus (HIV) drugs indicate a need for discovery and development of potent and safe antivirals with a novel mechanism of action. Maturation inhibitors (MIs) represent one such new class of HIV therapies. MIs inhibit a late step in the HIV-1 Gag processing cascade, causing defective core condensation and the release of non-infectious virus particles from infected cells, thus blocking the spread of the infection to new cells. Clinical proof-of-concept for the MIs was established with betulinic acid derived bevirimat, the prototype HIV-1 MI. Despite the discontinuation of its further clinical development in 2010 due to a lack of uniform patient response caused by naturally occurring drug resistance Gag polymorphisms, several second-generation MIs with improved activity against viruses exhibiting Gag polymorphism mediated resistance have been recently discovered and are under clinical evaluation in HIV/AID patients. In this review, current understanding of HIV-1 MIs is described and recent progress made toward elucidating the mechanism of action, target identification and development of second-generation MIs is reviewed.

Lack of an effective small-animal model to study the Kaposi's sarcoma-associated herpesvirus (KSHV) infection in vivo has hampered studies on the pathogenesis and transmission of KSHV. The objective of our study was to determine whether the humanized BLT (bone marrow, liver, and thymus) mouse (hu-BLT) model generated from NOD/SCID/IL2rγ mice can be a useful model for studying KSHV infection. We have tested KSHV infection of hu-BLT mice via various routes of infection, including oral and intravaginal routes, to mimic natural routes of transmission, with recombinant KSHV over a 1-or 3-mo period. Infection was determined by measuring viral DNA, latent and lytic viral transcripts and antigens in various tissues by PCR, in situ hybridization, and immunohistochemical staining. KSHV DNA, as well as both latent and lytic viral transcripts and proteins, were detected in various tissues, via various routes of infection. Using double-labeled immune-fluorescence confocal microscopy, we found that KSHV can establish infection in human B cells and macrophages. Our results demonstrate that KSHV can establish a robust infection in the hu-BLT mice, via different routes of infection, including the oral mucosa which is the most common natural route of infection. This hu-BLT mouse not only will be a useful model for studying the pathogenesis of KSHV in vivo but can potentially be used to study the routes and spread of viral infection in the infected host. T he Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as the human herpesvirus 8, was first identified from KS tissues in 1994 (1). It is the etiologic agent for KS and is also associated with primary effusion lymphoma (PEL) and multicentric Castleman's disease (2). More recently it was also found to be associated with KSHV-associated inflammatory cytokine syndrome (3). Although substantial progress has been made in characterizing the virus, there are still many unanswered questions such as how KSHV infection can lead to disease manifestation and whether latent or lytic induction of KSHV are associated with malignancies. One of the reasons is a lack of a good small-animal model to study KSHV infection in vivo, which has hampered studies on how KSHV infects, spreads, and how it interacts with the host and ultimately leads to disease pathogenesis. Moreover, currently there is no vaccine against KSHV infection, and there is need for an effective animal model to evaluate the efficacy of vaccines if they are developed and for the testing of antiviral regimens.An ideal model should have relatively short generation time, reproduce rapidly, be inexpensive to maintain and house, and be easy to manipulate. An example is a rodent model that can be infected by KSHV effectively. Several small-rodent models have been tested for KSHV infection. The models include transplantation with both human KSHV-infected B lymphoma cells and primary human peripheral blood mononuclear cells in the SCID mouse (4), injection of KSHV into the human skin engrafted or the transplant of the SCID mice...

Significant enhancement of photoluminescence in ALD Al2O3 passivated porous 6H-SiC.

3-O-(3,3-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.3-O-(3Ј,3Ј-Dimethylsuccinyl) betulinic acid (DSB), also known as bevirimat (BVM), is a potent inhibitor of HIV-1 maturation (7,8,20,21). BVM targets the HIV-1 Gag CA-SP1 boundary region by blocking viral protease cleavage of SP1 from the CA-SP1 precursor and inhibiting release of the mature CA protein, which is the final step required for virion maturation. Despite the novel mechanism of action and initial progress made in small-scale clinical trials (11-13, 17), further development of BVM has encountered unexpected challenges in the clinical setting, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not respond well to BVM treatment (3,10,14,16). These three residues (glutamine-valine-threonine [QVT]) are referred to as the SP1 polymorphism motif. Interestingly, these naturally occurring mutations that confer intrinsic resistance to BVM were not identified by in vitro drug resistance selection experiments and are not located in the region immediately flanking the CA-SP1 cleavage site (2,7,8). Extensive in vitro drug selection experiments identified six amino acid changes (proximal to the CA-SP1 cleavage site) that independently confer BVM resistance (2). Three substitutions were located at the 1st and 3rd residues of SP1 (A1V, A3V, and A3T), and three substitutions were identified at the extreme C terminus of CA (H226Y, L231M, and L231F).There are extensive data on HIV-1 subtype B viruses and their representative molecular clone pNL4-3 concerning BVM's mechanism of action and antiviral activity (2,4,7,8,20,21). In contrast, HIV-1 non-B subtypes and derived molecular clones have received less attention, despite knowledge that these non-B subtypes are responsible for nearly 90% of the current worldwide HIV-1 pandemic (6,19). Additionally, we noted that non-B subtypes exhibit more frequent changes in the identified SP1 polymorphism motif than B subtype viruses do. The...