Gag-Pol Processing during HIV-1 Virion Maturation: A Systems Biology Approach

Proteolytic processing of Gag and Gag-Pol polyproteins by the viral protease (PR) is crucial for the production of infectious HIV-1, and inhibitors of the viral PR are an integral part of current antiretroviral therapy. The process has several layers of complexity (multiple cleavage sites and substrates; multiple enzyme forms; PR auto-processing), which calls for a systems level approach to identify key vulnerabilities and optimal treatment strategies. Here we present the first full reaction kinetics model of proteolytic processing by HIV-1 PR, taking into account all canonical cleavage sites within Gag and Gag-Pol, intermediate products and enzyme forms, enzyme dimerization, the initial auto-cleavage of full-length Gag-Pol as well as self-cleavage of PR. The model allows us to identify the rate limiting step of virion maturation and the parameters with the strongest effect on maturation kinetics. Using the modelling framework, we predict interactions and compensatory potential between individual cleavage rates and drugs, characterize the time course of the process, explain the steep dose response curves associated with PR inhibitors and gain new insights into drug action. While the results of the model are subject to limitations arising from the simplifying assumptions used and from the uncertainties in the parameter estimates, the developed framework provides an extendable open-access platform to incorporate new data and hypotheses in the future.     

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6^gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3′ end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6^gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.The transframe protein p6*, also referred to as TFP-p6^pol, is one of the least-characterized gene products in human immunodeficiency virus type 1 (HIV-1), consistently raising controversial discussions on its main function in the viral life cycle. p6* is located at the amino terminus of the Pol moiety within the Gag-Pol precursor, which is synthesized following a programmed ribosomal −1 frameshift during translation (Fig. ​(Fig.1).1). In contrast, the Gag precursor is generated by conventional translation at a 20-fold-higher rate (summarized in reference 13). The characterization of recombinant p6* protein by nuclear magnetic resonance analysis revealed a highly flexible structure (3), suggesting that p6* might serve as an adjustable hinge within the large Gag-Pol precursor to facilitate folding of the bulky protein domains during viral assembly. Proper folding of the embedded homodimeric protease (PR) is an essential prerequisite for full activity following sequential autocleavage from the full-length Gag-Pol polyprotein (summarized in reference 13). Indeed, the p6* residues have been demonstrated to stabilize the PR dimer and dictate the folding propensities of PR precursors, thereby influencing the rate of PR maturation (6, 10, 21). Interestingly, the autoprocessing of a truncated Gag-PR precursor was clearly accelerated when the p6* sequence was deleted, leading to the proposal that the active site of the PR might be less accessible in the presence of p6* (26). Further observations that p6* itself is sequentially cleaved by the PR (1, 7, 22, 23, 24, 30, 31, 32, 41) strengthened the hypothesis that the transframe protein contributes to spatiotemporal activation of the PR. Indeed, we and others have previously shown that the PR needs to be released from the flanking p6* residues to be capable of completing all virus-associated processing steps (24, 27, 36). The potential of p6* to regulate PR activity has been further corroborated by our finding that recombinant p6* protein comprising a free accessible carboxyl terminus is a potent inhibitor of the mature PR in vitro (28). Apart from the carboxyl-terminal tetrapeptide mainly contributing to the observed inhibition, the amino-terminal octapeptide of p6* (TFP) has been reported to inhibit PR activity in vitro (21).Open in a separate windowFIG. 1.Gag and Gag-Pol polyproteins encoded by NL4-3 (NL) and AL proviruses. (A) Schematic representation of the Gag and Gag-Pol polyprotein precursors. The p6* domain within Gag-Pol is highlighted in black, and the p1-p6^gag region inserted in the Gag-Pol precursor of AL is shaded gray; the five residues appended to p6^gag in Gag are indicated by a black vertical line. MA, matrix protein; NC, nucleocapsid protein; IN, integrase. (B) The Gag (gray) and Pol (black) products translated in the presence of the original frameshift site in NL are indicated on top, and the corresponding passage synthesized in AL after destruction of the slippery site is shown below. The active (NL) and inactive (AL) slippery sites are underlined, and the 3′ nucleotides contributing to the stem-loop structure are in parentheses. (C) An artificial frameshift site was introduced at the 3′ end of the gag gene in AL, resulting in the synthesis of a Gag-Pol polyprotein with p6* directly fused to p6^gag. The gag reading frame in AL is terminated by an artificial stop codon (boxed) and is extended by the residues ANFLG. The E₄→V₄ substitution in p6* is marked by a circle, and numbers at the left indicate amino acid positions within Gag and Gag-Pol with respect to the Gag start codon.The fact that p6* has a substantial size of 55 to 72 amino acids, depending on the isolate, raises the question of whether the transframe protein exerts other functions apart from PR regulation. In this regard, p6* has also been proposed to be a possible binding partner of the viral Nef protein, the major pathogenicity factor during HIV infection (9). However, the target site within p6* for Nef interaction has not been mapped so far.Although it is completely overlapped by the p1-p6^gag domains, the p6* sequence offers much room for natural polymorphisms (5). Furthermore, amino acid insertions or duplications, as well as deletions of up to 13 residues, in p6* sequences of infectious isolates have been reported, some of which are associated with viral drug resistance (4, 29, 35, 38). We have recently shown that nonconservative substitutions of up to 70% of the p6* residues in a provirus clone did not abolish viral growth or infectivity in various cell lines, suggesting that the central p6* region is widely dispensable for viral in vitro replication (19, 27).Unlike the variable center, the p6* amino terminus is highly conserved owing to the overlapping p1^gag sequence (14) and the RNA frameshift signal composed of a heptanucleotide slippery site (UUUUUUA) and a 3′ pseudoknot structure (11). This complex sequence context imposes major restrictions on mutational analysis of the p6* amino terminus in the viral background (24). To allow for a more in-depth functional analysis of the conserved p6* residues in the viral context, we have established a novel NL4-3-based provirus with p1-p6^gag and p6* reading frames uncoupled via a dislocated frameshift site. Thereby, we could demonstrate that neither the deletion of the bulk of p6* nor the insertion of a short unrelated sequence significantly affected replication and infectivity of the virus in different cell cultures. However, the insertion of a five times larger green fluorescent protein (GFP)-encoding sequence into the p6* reading frame was associated with a clear loss of particle production and infectivity. Therefore, we conclude that p6* is a spacer protein of limited length which is widely dispensable for viral replication in vitro.     

Human Herpesvirus 6A U14 Is Important for Virus Maturation

Human herpesvirus 6A (HHV-6A) U14 is a virion protein with little known function in virus propagation. Here, we elucidated its function by constructing and analyzing U14-mutated viruses. We found that U14 is essential for HHV-6A propagation. We then constructed a mutant virus harboring dysfunctional U14. This virus showed severely reduced growth and retarded maturation. Taken together, these data indicate that U14 plays an important role during HHV-6A maturation.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

Brownian and Essential Dynamics Studies of the HIV-1 Integrase Catalytic Domain

Abstract

The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computional approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a “gating”-like motion with respect to the active site.     

Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.     

The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner

The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55(Gag). Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.     

Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice

Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.     

A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4

The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of Escherichia coli, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757–761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.     

Essential ingredients for HIV-1 budding

HIV-1 engages the cellular ESCRT-III/VPS4 membrane scission machinery for its escape from host cells. Three papers now begin to demystify its mode of action by showing that HIV-1 requires only the transient recruitment of a surprisingly small subset of ESCRT-III components, whose membrane abscission function depends on VPS4 activity.     

The Heptad Repeat 2 Domain Is a Major Determinant for Enhanced Human Immunodeficiency Virus Type 1 (HIV-1) Fusion and Pathogenicity of a Highly Pathogenic HIV-1 Env

Human immunodeficiency virus type 1 (HIV-1)-mediated depletion of CD4⁺ lymphocytes in an infected individual is the hallmark of progression to AIDS. However, the mechanism for this depletion remains unclear. To identify mechanisms of HIV-1-mediated CD4 T-cell death, two similar viral isolates obtained from a rapid progressor patient with significantly different pathogenic phenotypes were studied. One isolate (R3A) demonstrates enhanced pathogenesis in both in vivo models and relevant ex vivo lymphoid organ model systems compared to another isolate, R3B. The pathogenic determinants were previously mapped to the V5-gp41 envelope region, correlating functionally with enhanced fusion activity and elevated CXCR4 binding affinity. To further elucidate specific differences between R3A and R3B within the V5-gp41 domains that enhance CD4 depletion, R3A-R3B chimeras to study the V5-gp41 region were developed. Our data demonstrate that six residues in the ectodomain of R3A provide the major determinant for both enhanced Env-cell fusion and pathogenicity. Furthermore, three amino acid differences in the heptad repeat 2 (HR-2) domain of R3A determined its fusion activity and significantly elevated its pathogenic activity. The chimeric viruses with enhanced fusion activity, but not elevated CXCR4 affinity, correlated with high pathogenicity in the thymus organ. We conclude that the functional domain of a highly pathogenic HIV-1 Env is determined by mutations in the HR-2 region that contribute to enhanced fusion and CD4 T-cell depletion.Human immunodeficiency virus type 1 (HIV-1) is the causative agent for AIDS, which is characterized by a dramatic loss of CD4⁺ lymphocytes and impairment of the immune system against invading pathogens (13, 21, 22). Though much has been determined regarding interactions between HIV-1 virus and CD4⁺ target cells, the mechanisms by which the HIV-1 virus depletes CD4⁺ lymphocytes remain incompletely understood. Various studies have demonstrated that in an HIV-infected host, both infected and uninfected cells are prone to destruction, albeit by different pathways (15, 18, 29). Recently, our group and others have shown that while binding of CD4 and chemokine receptors contribute to syncytium formation in vitro, viral membrane fusion by the envelope glycoprotein plays an important role in depletion of both uninfected and infected cells by HIV-1 and simian-human immunodeficiency virus in vivo (1, 11, 12, 26, 29).HIV-1 entry into a cell is mediated by a multistep process that begins with high-affinity binding between viral envelope (gp120) and the cellular CD4 receptor (9, 14, 16). This binding causes a conformational change in the viral envelope, allowing for subsequent coreceptor binding (mainly CCR5 or CXCR4). Upon coreceptor binding, another conformational change is thought to take place that allows gp41 to engage the cell to form a fusion complex. Envelope proteins have been demonstrated to exist as a trimer, allowing for three gp41s to form a fusion assembly through noncovalent interactions. This fusion assembly is determined to exist in a six-helix bundle formation as the fusion event takes place, allowing for the virion to fuse to the host cell (5, 24).The envelope glycoprotein (Env) of HIV plays a significant role in viral pathogenesis, as seen in several in vitro and in vivo models of infection. The Env functions to mediate virus entry of cells and is also a major target for immune responses (31, 39). While the envelope initially forms as a precursor protein (gp160), subsequent cleavage by a cellular protease yields the surface subunit gp120 and the transmembrane gp41 although the gp120 and gp41 interact noncovalently (36). The gp120 protein is comprised of five variable (V1 to V5) and five conserved constant (C1 to C5) domains and binds CD4 and the coreceptors. The gp41 protein is comprised of an amino-terminal fusion domain and two heptad repeats (HR-1 and HR-2) in the ectodomain (extracellular domain), a single transmembrane domain, and a cytoplasmic tail (intracellular domain) (8, 10, 36, 37). Due to the discovery of fusion inhibitor peptides such as C34 (23, 24) and T20 (38), much is now known about the fusion complex formed by the HIV-1 fusion domain. Similar to other viral envelopes that carry a type 1 fusion complex (such as influenza and corona viruses), the ectodomain of HIV-1 Env carries two HRs that form a coiled-coiled structure. In order for HIV-cell fusion to occur, the HR-1 domains of the trimeric Env protein must interact with the cell surface. Following this initial interaction, HR-2 domains are thought to intertwine over the HR-1 coils to form a stable six-helix bundle, which represents the gp41 core structure. X-ray crystallographic studies show that the six-helix bundle core consists of the HR-1 and HR-2 peptides bound in an antiparallel manner. This structure brings the fusion peptide to the target cell membrane, allowing for the formation of a fusion pore and the entry of virions into the cell.HIV-1 Env expressed on the surface of infected cells can induce cell-cell fusion with adjacent uninfected cells to form multinucleated syncytia and single cell lysis in cell culture and apoptosis in primary cells. Various models (both ex vivo and in vivo) have been utilized to study HIV-1-induced depletion of CD4⁺ lymphocytes. Models such as SCID-human thymus-liver (SCID-hu thy/liv), tonsil histoculture, and human fetal thymus organ culture (HFTOC) have demonstrated significant use in the study of acute infection and pathogenesis in the appropriate lymphoid organ microenvironment as they retain the organ structure and do not require exogenous stimulation for productive viral infection to occur (2, 20, 28, 32). More importantly, tissue culture-adapted HIV-1 isolates such as HXB2 fail to replicate in the SCID-hu thy/liv or HFTOC models (30, 33). Organ models such as the SCID-hu thy/liv and HFTOC thus more accurately demonstrate infection, replication, and pathogenicity of primary HIV-1 strains.Here, HFTOC is used to investigate mechanisms by which an HIV-1 virus with a highly pathogenic viral Env is able to deplete CD4⁺ lymphocytes. Two viral isolates obtained from rapid progressor patient 3 of the ALIVE cohort (40) show significant sequence homology, particularly in the Env region, while they carry stark differences in pathogenic ability (26, 27). One isolate (denoted R3A) was found to demonstrate enhanced fusion in cell-cell fusion assays as well as enhanced pathogenesis in relevant ex-vivo/in vivo organ model systems compared to another isolate, R3B. To define the pathogenic determinants that differentiate R3A from R3B, this study demonstrates that the enhanced fusogenicity of R3A (governed by the ectodomain of the gp41), but not the elevated CXCR4 binding affinity, confers the pathogenic phenotype in HFTOC. We further demonstrate that three amino acid differences in the HR-2 domain allow for this enhanced fusion for R3A Env, defining a possible mechanism for a pathogenic HIV-1 envelope.     

The Carboxy-Terminal Domain of ROS1 Is Essential for 5-Methylcytosine DNA Glycosylase Activity

Arabidopsis thaliana repressor of silencing 1 (ROS1) is a multi-domain bifunctional DNA glycosylase/lyase, which excises 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as well as thymine and 5-hydroxymethyluracil (i.e., the deamination products of 5mC and 5hmC) when paired with a guanine, leaving an apyrimidinic (AP) site that is subsequently incised by the lyase activity. ROS1 is slow in base excision and fast in AP lyase activity, indicating that the recognition of pyrimidine modifications might be a rate-limiting step. In the C-terminal half, the enzyme harbors a helix–hairpin–helix DNA glycosylase domain followed by a unique C-terminal domain. We show that the isolated glycosylase domain is inactive for base excision but retains partial AP lyase activity. Addition of the C-terminal domain restores the base excision activity and increases the AP lyase activity as well. Furthermore, the two domains remain tightly associated and can be co-purified by chromatography. We suggest that the C-terminal domain of ROS1 is indispensable for the 5mC DNA glycosylase activity of ROS1.     

The Y-S-L-I Tyrosine-Based Motif in the Cytoplasmic Domain of the Human T-Cell Leukemia Virus Type 1 Envelope Is Essential for Cell-to-Cell Transmission

The human T-cell leukemia virus type 1 (HTLV-1) transmembrane glycoprotein has a 24-amino-acid cytoplasmic domain whose function in the viral life cycle is poorly understood. We introduced premature-stop mutations and 18 single-amino-acid substitutions into this domain and studied their effects on cell-to-cell transmission of the virus. The results show that the cytoplasmic domain is absolutely required for cell-to-cell transmission of HTLV-1, through amino acids which cluster in a Y-S-L-I tyrosine-based motif. The transmission defect in two motif mutants did not result from a defect in glycoprotein incorporation or fusion. It appears that the Y-S-L-I tyrosine-based motif of the HTLV-1 glycoprotein cytoplasmic domain has multiple functions, including involvement in virus transmission at a postfusion step.     

AKAP9 Is Essential for Spermatogenesis and Sertoli Cell Maturation in Mice

Mammalian male fertility relies on complex inter- and intracellular signaling during spermatogenesis. Here we describe three alleles of the widely expressed A-kinase anchoring protein 9 (Akap9) gene, all of which cause gametogenic failure and infertility in the absence of marked somatic phenotypes. Akap9 disruption does not affect spindle nucleation or progression of prophase I of meiosis but does inhibit maturation of Sertoli cells, which continue to express the immaturity markers anti-Mullerian hormone and thyroid hormone receptor alpha in adults and fail to express the maturation marker p27^Kip1. Furthermore, gap and tight junctions essential for blood–testis barrier (BTB) organization are disrupted. Connexin43 (Cx43) and zona occludens-1 are improperly localized in Akap9 mutant testes, and Cx43 fails to compartmentalize germ cells near the BTB. These results identify and support a novel reproductive tissue-specific role for Akap9 in the coordinated regulation of Sertoli cells in the testis.