African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information. African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.

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African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ϳ2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T ‫؍‬ 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T ‫؍‬ 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it.

The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide +1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ alpha forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template-primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template-primers. Altogether, these data suggest that Val75 interactions with the 5' template overhang are important determinants of fidelity.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication. IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3-azido-3-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA⅐DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.Human immunodeficiency virus type 1 (HIV-1) 1 reverse transcriptase (RT) replicates the viral genomic RNA to synthesize a double-stranded DNA that integrates into the host genome. The viral enzyme is multifunctional, possessing RNAand DNA-dependent DNA polymerase, RNase H, strand transfer, and strand displacement activities (1). HIV-1 RT is a heterodimeric enzyme composed of two polypeptide chains of 66 and 51 kDa, with subdomains termed fingers, thumb, palm, and connection in both subunits and an RNase H domain in the larger subunit only.HIV-1 RT is an important target for chemotherapeutic intervention in the control of AIDS. Antiretroviral drugs targeting the viral polymerase include nucleoside analogue inhibitors (NRTIs), acyclic nucleoside phosphonates, and non-nucleoside RT inhibitors (NNRTIs) (reviewed in Ref. 2). Inside the cell, nucleoside derivatives are converted to their active triphosphate forms by host cell kinases and are then incorporated into the HIV-1 genome by the viral RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation blocks elongation of the growing DNA chain. On the other hand, NNRTIs bind to an allosteric site located 10 -15 Å away from the polymerase active site, distorting the geometry and/or the mobility...

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