Structure of the St. Louis encephalitis virus postfusion envelope trimer

St. Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest virus, an alphavirus. Domains II and III (DII and DIII) of SLEV E also adopt different angles relative to DI, which suggests that the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved among flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variations in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis

Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10−2 to 10−5 in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (∼10−4 to 10−5). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Δvif efficiently

Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses

Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3G In Vivo

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.United States. Public Health Service (Grant R01-AI-085015)United States. Public Health Service (Grant T32-CA115299 )United States. Public Health Service (Grant F32-AI100512

Evolution of the Primate APOBEC3A Cytidine Deaminase Gene and Identification of Related Coding Regions

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1–4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme

The Restriction of Zoonotic PERV Transmission by Human APOBEC3G

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections

APOBEC3G and APOBEC3F Require an Endogenous Cofactor to Block HIV-1 Replication

APOBEC3G (A3G)/APOBEC3F (A3F) are two members of APOBEC3 cytidine deaminase subfamily. Although they potently inhibit the replication of vif-deficient HIV-1, this mechanism is still poorly understood. Initially, A3G/A3F were thought to catalyze C-to-U transitions on the minus-strand viral cDNAs during reverse transcription to disrupt the viral life cycle. Recently, it was found more likely that A3G/A3F directly interrupts viral reverse transcription or integration. In addition, A3G/A3F are both found in the high-molecular-mass complex in immortalized cell lines, where they interact with a number of different cellular proteins. However, there has been no evidence to prove that these interactions are required for A3G/A3F function. Here, we studied A3G/A3F-restricted HIV-1 replication in six different human T cell lines by infecting them with wild-type or vif-deficient HIV-1. Interestingly, in a CEM-derived cell line CEM-T4, which expresses high levels of A3G/A3F proteins, the vif-deficient virus replicated as equally well as the wild-type virus, suggesting that these endogenous antiretroviral genes lost anti-HIV activities. It was confirmed that these A3G/A3F genes do not contain any mutation and are functionally normal. Consistently, overexpression of exogenous A3G/A3F in CEM-T4 cells still failed to restore their anti-HIV activities. However, this activity could be restored if CEM-T4 cells were fused to 293T cells to form heterokaryons. These results demonstrate that CEM-T4 cells lack a cellular cofactor, which is critical for A3G/A3F anti-HIV activity. We propose that a further study of this novel factor will provide another strategy for a complete understanding of the A3G/A3F antiretroviral mechanism

APOBEC3A Is a Specific Inhibitor of the Early Phases of HIV-1 Infection in Myeloid Cells

Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for viral spread and as a viral reservoir. Yet, cells of this lineage generally resist HIV-1 infection when compared to cells of other lineages, a phenomenon particularly acute during the early phases of infection. Here, we explore the role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that is highly expressed in myeloid cells, but so far lacks a known antiviral effect against retroviruses. Using ectopic expression of APOBEC3A in established cell lines and specific silencing in primary macrophages and dendritic cells, we demonstrate that the pool of APOBEC3A in target cells inhibits the early phases of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1, specifically in cells of myeloid origins. In these cells, APOBEC3A affects the amount of vDNA synthesized over the course of infection. The susceptibility to the antiviral effect of APOBEC3A is conserved among primate lentiviruses, although the viral protein Vpx coded by members of the SIVSM/HIV-2 lineage provides partial protection from APOBEC3A during infection. Our results indicate that APOBEC3A is a previously unrecognized antiviral factor that targets primate lentiviruses specifically in myeloid cells and that acts during the early phases of infection directly in target cells. The findings presented here open up new venues on the role of APOBEC3A during HIV infection and pathogenesis, on the role of the cellular context in the regulation of the antiviral activities of members of the APOBEC3 family and more generally on the natural functions of APOBEC3A

Human cellular restriction factors that target HIV-1 replication

Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G), bone marrow stromal cell antigen 2 (BST-2), cyclophilin A, tripartite motif protein 5 alpha (Trim5α), and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions