A-type Inclusion Research Articles

Zoonotic Infections of Buffalopox in India

Four outbreaks of buffalopox in domestic buffaloes, with considerable mortality with high case fatality rates in young buffalo calves and high morbidity with significant productivity loss in terms of reduction in milk yield in adult animals along with severe zoonotic infection in milk attendants were recorded at various places in India, during 2006-2008. In buffaloes, the pox lesions were confined to udder and teats of the majority of the affected animals, and in few animals the lesions were appeared on the hindquarters, indicating generalized infection. The overall disease morbidity, mortality and case fatality rate were 6.8%, 0.7% and 11.4% respectively. Milkers developed pox-like lesions on the hands, forearms and forehead accompanied by fever, axillary lymphadenopathy and general malaise. The causative agent of the outbreaks, buffalopox virus (BPXV), was confirmed upon virus isolation in cell culture, electron microscopy, A-type inclusion (ATI) and ankyrin repeat protein (C18L) gene-specific polymerase chain reactions (PCR). Further, sequence analysis of the BPXV isolates from human and buffalo showed more identity of ATI and C18L genes sequences with that of other orthopoxviruses at nucleotide and amino acid levels and confirmed a close relationship of BPXV with Vaccinia virus (VACV) or VACV-like viruses. Considering the zoonotic impact and productivity losses of buffalopox infection, the control measures are imperative in curtailing economic and public health impact of the disease.

Comparative whole genome sequence analysis of wild-type and cidofovir-resistant monkeypoxvirus

We performed whole genome sequencing of a cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl) cytosine] [HPMPC]}-resistant (CDV-R) strain of Monkeypoxvirus (MPV). Whole-genome comparison with the wild-type (WT) strain revealed 55 single-nucleotide polymorphisms (SNPs) and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV). These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV) species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies.

Congregation of Orthopoxvirus Virions in Cytoplasmic A-Type Inclusions Is Mediated by Interactions of a Bridging Protein (A26p) with a Matrix Protein (ATIp) and a Virion Membrane-Associated Protein (A27p)

Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.

Isolation and characterization of Indian isolates of camel pox virus

In this study, we isolated and identified three camel pox viruses (CMLV) from two outbreaks of camel pox infection in camels associated with eruptions on cheeks, nostrils, limbs, scrotum, and sheath that occurred at different places of Bikaner district, Rajasthan (India). The scab specimens collected were subjected for virus isolation in Vero cell culture, and the isolated viruses were characterized by employing polymerase chain reaction (PCR) and sequencing. The causative agent was identified as CMLV, based on A-type inclusion, B5R and C18L genes-specific PCRs and partial sequencing of these genes, which clearly confirmed that the outbreaks were caused by CMLV and identity of CMLV isolates. Further, phylogenetic analysis of partial C18L gene sequences have showed that Indian CMLV are clustered together with other reported isolates/strains.

An Outbreak of Orthopoxvirus-Associated Disease in Horses in Southern Brazil

An outbreak of severe cutaneous disease associated with an orthopoxvirus in horses in southern Brazil is described. Fourteen Crioulo mares and foals from a husbandry farm developed papules, and vesicles progressing to proliferative and exudative lesions on the muzzle, external nares, and external and internal lips. The vesicles eroded, and the proliferative lesions eventually bled and progressed to moist crusts and scars. The clinical signs lasted approximately 6-12 days, after which the animals progressively recovered. Direct electron microscopy of skin biopsies revealed brick-shaped, 250-300-nm virus particles with orthopoxvirus morphology. Histological examination of the lesions revealed vacuolar degeneration of the cells of the stratum spinosum and the presence of large intracytoplasmic, eosinophilic inclusion bodies. Polymerase chain reaction amplification of poxvirus A-type inclusion body gene confirmed the presence of orthopoxvirus DNA in horse tissues. Inoculation of tissue homogenates into the chorioallantoic membrane of chicken embryonated eggs and intraperitoneally in mice resulted in pock-like lesions with the microscopic appearance of poxvirus-induced histopathology. Taken together, these results demonstrate the association of an orthopoxvirus with the outbreak of cutaneous disease in horses. The origin of the agent causing the outbreak is uncertain because no similar condition has been reported in Brazil.

Partial genetic characterization of viruses isolated from pox-like infection in cattle and buffaloes: evidence of buffalo pox virus circulation in Indian cows

Characterization of field isolates of viruses associated with pox-like outbreaks involving both cows (cattle) and buffaloes was carried out. PCR and electron microcopy of representative virus isolates from these animals, initially identified them as orthopoxviruses (OPXVs). Sequence and phylogenetic analyses of A-type inclusion and haemagglutinin (HA) genes of these isolates revealed a closer relationship with other OPXVs. Sequencing of the HA gene of these isolates revealed sequence identity of 96.2-99.8 and 94.6-98.7% at the nucleotide and deduced amino acid level, respectively, with VACVs, particularly with VACV-MVA, a vaccine strain. Further, C18L (ankyrin repeat protein)-gene-based BPXV-specific PCR confirmed them as BPXV. It is apparent from this study that pox-like outbreaks in cows and buffaloes in India are caused, in most cases, by BPXV. Considering the zoonotic implications of buffalopox, such outbreaks involving both buffaloes and cows in a mixed flock may pose a significant public health threat. Transmissibility of BPXV between different species including cows, buffaloes and human beings implies the potential reemergence of the virus in the subcontinent, similar to vaccinia-like outbreaks witnessed recently in other countries.

Comparative sequence analysis of A-type inclusion (ATI) and P4c proteins of orthopoxviruses that produce typical and atypical ATI phenotypes

Some orthopoxviruses produce large proteinaceous intracellular bodies, known as A-type inclusions (ATIs) during infection of host cells. Virions associate with ATIs resulting in distinct phenotypes referred to as V+, V+/ and V⁻. The phenotype V+ has the virions embedded in the ATI matrix; V⁻ has no virions embedded within or on the surface of the ATI matrix, whereas an aberrant phenotype, the V+/ has virions only on the surface of ATIs. Viruses that do not produce ATI are designated as V⁰. Recombinant viruses generated from a V+ cowpox virus (CPXV) and a V⁰ transgenic vaccinia virus (VACV) produced aberrant V+/ ATIs. ATI phenotype is dependent on the A-type inclusion protein (Atip) and the P4c protein. We sequenced the atip and p4c genes of parental and progeny recombinant viruses as well as their flanking sequences. The atip and p4c open reading frames were identical in parental V+ CPXV and hybrid V+/ progenies. Our results suggest that additional viral gene(s) are required for the formation of wild type V+ ATI.

Molecular simulation of β-cyclodextrin inclusion complex with 2-phenylethyl alcohol

The structures of β-cyclodextrin inclusion complexes with 2-phenylethyl alcohol in vacuum and aqueous solution have been investigated by using molecular dynamics simulation. The inclusion structures and the physicochemical stability of the complexes were also analysed, discussed and validated by ultraviolet spectrums and thermodynamic properties. The results of molecular dynamics simulation indicate that the A-type β-cyclodextrin inclusion complex with 2-phenylethyl alcohol in both vacuum and aqueous solution have better physical stability, and its chemical stability also has obvious promotion than that of free one. Therefore, the β-cyclodextrin can be used to control and regulate the release of the 2-phenylethyl in food.

Vaccinia virus A26 and A27 proteins form a stable complex tethered to mature virions by association with the A17 transmembrane protein.

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

Diagnosis and Assessment of Monkeypox Virus (MPXV) Infection by Quantitative PCR Assay: Differentiation of Congo Basin and West African MPXV Strains

Human monkeypox, an infectious disease caused by monkeypox virus (MPXV), is endemic to western and central Africa. A LightCycler quantitative PCR (LC-qPCR) system was developed for the diagnosis of this disease, targeting the A-type inclusion body gene (ATI gene) of MPXV. One naive monkey was infected with MPXV Zr-599 (Congo Basin strain) and one with MPXV Liberia (West African strain). Another three monkeys were immunized with smallpox vaccine on 0, 3, or 7 days, respectively, before infection with MPXV Zr-599. Peripheral blood cell (PBC) and throat swab (TS) specimens were serially collected. The LC-qPCR was validated for the diagnosis of monkeypox using virus isolation. Sequencing of the partial ATI gene revealed the insertion of a unique 453-nucleotide residue in the West African strains but not in the Congo Basin strains. Specific reverse primers for Congo Basin and West African strains were designed based on the unique sequence insertion. The LC-qPCR detected the MPXV genome, but not those of the other orthopoxviruses tested nor the varicella-zoster virus. Both the sensitivity and specificity of the LC-qPCR were over 90% in comparison to virus isolation when TS specimens were tested. Fourteen of the 15 virus isolation-positive PBC specimens showed positive reactions in the assay. Further, most PBC specimens collected from symptomatic monkeys in the later stage of illness showed positive reactions in the assay but negative reaction in virus isolation. It was possible to differentiate between these two groups with the LC-qPCR. Thus, the newly developed LC-qPCR is a useful and reliable diagnostic tool for MPXV infection.

Purification and Properties of the Vaccinia Virus mRNA Processing Factor

The mRNAs encoding the vaccinia virus F17 protein and the cowpox A-type inclusion protein are known to possess sequence-homogeneous 3' ends, generated by a post-transcriptional cleavage event. By using partially purified extracts, we have previously shown that the same factor probably cleaves both the F17 and A-type inclusion protein transcripts and that the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. In this study, we have purified the cleavage factor from vaccinia-infected HeLa cells using column chromatography and gel filtration. The factor eluted from the gel filtration column with an apparent molecular mass of approximately 440 kDa. Mass spectrometric analyses of the proteins present in the peak active fractions revealed the presence of at least one vaccinia protein with a high degree of certainty, the H5R gene product. To extend this finding, extracts were prepared from HeLa cells infected with vaccinia virus overexpressing His-tagged H5, chromatographed on a nickel affinity column, and eluted using an imidazole gradient. Cleavage activity eluted with the peak of His-tagged H5. Gel filtration of the affinity-purified material further demonstrated that cleavage activity and His-tagged H5 co-chromatographed with an apparent molecular mass of 463 kDa. We therefore conclude that H5 is specifically associated with post-transcriptional cleavage of F17R transcripts. In addition, we show that dephosphorylation of a cleavage competent extract with a nonspecific phosphatase abolishes cleavage activity implying a role for phosphorylation in cleavage activity.

An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein

BackgroundProfilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins.ResultsProfilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed.ConclusionThese results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.

Proteome‐wide analysis of the serological response to vaccinia and smallpox

The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax, and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A-type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross-reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola-infected individuals and Dryvax vaccinees were indistinguishable. This is the first description of antibody-specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.

Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein. Since new-generation smallpox vaccines such as MVA will require extensive comparison to traditional smallpox vaccines in animal models of immunogenicity and protection, we compared the vaccinia virus antigens recognized by VIG to those recognized by sera from Dryvax and MVA immunized mice. The humoral immune response in immunized mice is qualitatively similar to that in humans.

Belo Horizonte virus: a vaccinia-like virus lacking the A-type inclusion body gene isolated from infected mice.

Here is described the isolation of a naturally occurring A-type inclusion body (ATI)-negative vaccinia-like virus, Belo Horizonte virus (VBH), obtained from a mousepox-like outbreak in Brazil. The isolated virus was identified and characterized as an orthopoxvirus by conventional methods. Molecular characterization of the virus was done by DNA cross-hybridization using Vaccinia virus (VACV) DNA. In addition, conserved orthopoxvirus genes such as vaccinia growth factor, thymidine kinase and haemagglutinin were amplified by PCR and sequenced. All sequences presented high similarity to VACV genes. Based on the sequences, phenograms were constructed for comparison with other poxviruses; VBH clustered consistently with VACV strains. Attempts to amplify the ATI gene (ati) by PCR, currently used to identify orthopoxviruses, were unsuccessful. Results presented here suggest that most of the ati gene is deleted in the VBH genome.

Sequence-independent acylation of the vaccinia virus A-type inclusion protein.

N-Terminal myristoylation of proteins typically occurs cotranslationally via an amide bond to the penultimate glycine residue within the canonical motif (M)GXXX(S/T/A) in a reaction catalyzed by N-myristoyltransferase. A second, less common myristoylation reaction occurs internally at dibasic amino acid doublets of proteins such as alpha-TNF. In this case, myristoylation occurs within a portion of the preprotein, which is subsequently removed by N-terminal proteolysis. The identity of the enzyme catalyzing internal myristoylation is unknown. Considering this information, the vaccinia virus (VV) A-type inclusion protein (ATI) presents a conundrum. Although this cytosolic protein is clearly myristoylated, the protein does not have the N-terminal myristoylation motif nor is it subject to proteolytic maturation. In the experiments reported here, we cleaved VV ATI with cyanogen bromide and determined that the myristoyl moiety was present in the C-terminal half of the protein. We also subjected a tryptic digest of VV ATI to liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry analyses, which indicated that ATI is randomly myristoylated at six different lysines or arginines. Analysis of the modification sites reveals no obvious conserved acceptor motifs or dibasic doublets. Mutation of these residues alone or in combination does not abrogate myristoylation of the protein, suggesting utilization of alternative modification sites. This information implies that the VV ATI protein is myristoylated in a sequence-independent manner. Because viral acylproteins typically utilize the host cell modification apparatus, this result suggests there may be an alternative type of myristoylation pathway in mammalian cells.

Post-transcription cleavage generates the 3′ end of F17R transcripts in vaccinia virus

Most vaccinia virus intermediate and late mRNAs possess 3′ ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3′ ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3′ end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3′ end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3′ end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor.

Identification and characterization of three immunodominant structural proteins of fowlpox virus.

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.

Characterization of a vaccinia-like virus isolated in a Brazilian forest.

The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.

Novel acylation of poxvirus A-type inclusion proteins

Myristylation is one of several post-translational modifications that occur on vaccinia virus (VV) proteins. Previously, time course labeling of VV-infected cells with myristic acid had indicated that five late proteins (17, 25, 36, 38 and 92 kDa) are myristylated. Four of these proteins were mapped to the E7R, L1R, AI6L and G9R open-reading frames, respectively, because of the predicted presence of the N-myristyltransferase recognition sequence (M-G-X-X-X-S/T/A) at their amino termini. In contrast, computer analyses of large (80–100 kDa) VV open reading frames did not reveal any predicted species with this N-terminal motif. By immunoprecipitation with monospecific sera and transient expression of cloned gene products, the myristylated 92-kDa protein has been demonstrated to be the A-type inclusion protein encoded by the Western Reserve (WR) strain of VV. Labeling of cowpox virus (CPV) infected cells with myristic acid indicated that the 160-kDa A-type inclusion protein appears to be myristylated as well. Both the VV 92-kDa and the CPV 160-kDa A-type inclusion proteins labeled with myristic acid were stable to hydroxylamine treatment, suggesting an amide linkage between the fatty acid and the acceptor protein. HPLC analysis confirmed that the 92-kDa protein was in fact myristylated. This data suggests that poxvirus ATI proteins may be subject to a novel type of internal myristylation modification, and the roles such modifications may play in the replication cycles of these viruses is discussed.