Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Mar 15;372(2):372-83.
doi: 10.1016/j.virol.2007.10.023. Epub 2007 Dec 3.

Identification from diverse mammalian poxviruses of host-range regulatory genes functioning equivalently to vaccinia virus C7L

Xiangzhi Meng  1 , Jie Chao, Yan Xiang
Affiliations

Identification from diverse mammalian poxviruses of host-range regulatory genes functioning equivalently to vaccinia virus C7L

Xiangzhi Meng et al. Virology. .

Abstract

Vaccinia virus (VACV) C7L is a host-range gene that regulates cellular tropism of VACV. Distantly related C7L homologues are encoded by nearly all mammalian poxviruses, but whether they are host-range genes functioning similar to VACV C7L has not been determined. Here, we used VACV as a model system to analyze five different C7L homologues from diverse mammalian poxviruses for their abilities to regulate poxvirus cellular tropism. Three C7L homologues (myxoma virus M63R, M64R and cowpox virus 020), when expressed with an epitope tag and from a VACV mutant lacking the host-range genes K1L and C7L (vK1L-C7L-), failed to support productive viral replication in human and murine cells. In nonpermissive cells, these viruses did not synthesize viral late proteins, expressed a reduced level of the early protein E3L, and were defective at suppressing cellular PKR activation. In contrast, two other C7L homologues, myxoma virus (MYXV) M62R and yaba-like disease virus (YLDV) 67R, when expressed with an epitope tag and from vK1L(-)C7L(-), supported normal viral replication in human and murine cells and restored the ability of the virus to suppress PKR activation. Furthermore, M62R rescued the defect of vK1L(-)C7L(-) at replicating and disseminating in mice following intranasal inoculation. These results show that MYXV M62R and YLDV 67R function equivalently to C7L at supporting VACV replication in mammalian hosts and suggest that a C7L-like host-range gene is essential for the replication of many mammalian poxviruses in mammalian hosts.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A multiple-sequence alignment of C7L and its homologues from mammalian poxviruses. They are VACV (vaccinia virus, orthopoxvirus) C7L, MYXV (myxoma virus, leporipoxvirus) M62R, YLDV (Yaba-like diseases virus, yatapoxvirus) 67R, SPPV (sheeppox virus, capripoxvirus) 063, SWPV (swinepox virus, suipoxvirus) 064, DPV (deerpox virus, unclassified) 076, MYXV M63R, MYXV M64R and CWPX (cowpox virus, orthopoxvirus) 020. Amino acids that are identical or similar in five or more of the nine proteins are highlighted. The dark and light shading indicate amino acids that are identical or similar, respectively.
Figure 2
Figure 2
Construction of recombinant VACV with its K1L deleted and its C7L substituted with a C7L homologue. (A). Schematic illustration of the construction and genomic organization of the recombinant VACV. The construction of K1L and C7L deletion mutant vK1L−C7L− was described previously (15). Recombinant VACV encoding the C7L homologue were constructed by homologous recombination of vK1L−C7L− and a plasmid encoding the C7L homologue and the green fluorescence protein (GFP). P11 indicates the VACV promoter that normally regulates late expression of the gene encoding the 11K protein (WR F18R). (B & C). The recombinant VACV expresses the expected C7L homologue. VERO cells were infected at a MOI of 10 PFU (B) or 25 PFU (C) per cell with the indicated viruses, which are referred by the name of ORF present at the C7L locus. The C7L homologue is tagged at its C-terminus with either a V5 or V5-TAP tag. At 8 hpi, the level of C7L homologue in infected cells was determined by Western blot with a monoclonal antibody (mAb) against V5. A longer exposure of the blot was shown on the right to illustrate the weaker band. On the same blot, the levels of VACV early protein E3L and host cell protein Hsp70 were also determined with an anti-E3L polyclonal antiserum and an anti-Hsp70 mAb. Hsp70 serves as a control for gel loading, while E3L serves as an indicator of VACV early protein expression. The size of the molecular weight marker in kilodaltons is shown on the left. The predicted molecular weights of the tagged C7L-homologues are listed on the right.
Figure 3
Figure 3
YLDV 67R and MYXV M62R can substitute C7L for its function in human and murine cell lines while CPXV 020, MYXV M63R and M64R cannot. (A). Plaque morphology of the recombinant VACV on representative cell lines. VERO, 3T3, HeLa and A431 cells were infected at a MOI of 0.01 PFU/cell with the indicated viruses. At approximately 36 hpi, GFP-expressing cells were visualized with an inverted fluorescence microscope using a 10× objective and photographed. (B). Growth curves of the recombinant VACV in HeLa cells at MOI of 5 PFU/cell. Virus yields at 0, 24 and 48 hpi were determined by plaque assay on the permissive VERO cells. The growth curves of the recombinant VACV in 3T3 and A431 were similar to the ones in HeLa cells and thus not shown.
Figure 4
Figure 4
vMYXV-M63R, vMYXV-M64R and vCWPX-020 are defective at viral late protein synthesis in nonpermissive HeLa cells. HeLa cells were infected with the indicated viruses at MOI of 10 PFU/cell. At 8 hpi, the level of viral early proteins C7L (or its homologue) and E3L were determined by Western blot as described in Fig 2B. At 12 hpi, the level of a VACV late protein encoded by ORF WR148 (11) was determined by Western blot with a mAb against WR148. WR148 is a remnant of orthopoxvirus Atype inclusion (ATI) protein.
Figure 5
Figure 5
The temporal expression of viral proteins by vVACV-C7L and vMYXV-M64R in VERO and HeLa cells. VERO (A) and HeLa cells (B) were infected at a MOI of 10 PFU per cell with vVACV-C7L or vMYXV-M64R. At the indicated time after infection (hpi), the level of C7L or M64R proteins was determined by Western blot with an anti-V5 antibody. The levels of VACV proteins E3L, WR148 and host cell protein Hsp70 were also determined by Western blot as respective control for VACV early, late protein expression and gel loading.
Figure 6
Figure 6
The defect of the host-range restricted viruses at suppressing cellular PKR and eIF2-α phosphorylation in HeLa cells correlates with a defect of the viruses at maintaining E3L expression. (A). The same cell lysates as described in Fig 5B were used to determine the levels of total and phosphorylated PKR and eIF2-α proteins in HeLa cells that were infected with vVACV-C7L or vMYXV-M64R for various time. The level of phosphorylated PKR and eIF2-α proteins were determined by Western blot with mAbs against phospho-PKR (Thr446) and phospho-eIF2α (Ser51). The blot was then stripped, and the levels of total PKR and eIF2-α were determined by Western blot with a mAb against total PKR and a polyclonal antibody against total eIF2-α. (B). HeLa cells were infected with the indicated viruses for 4 h and the level of total and phosphorylated PKR and VACV E3L proteins were determined by Western blot. (C). The same samples as shown in B were analyzed by Western blot with a multiplex of antibodies, including antibodies against phospho-p90RSK (Ser-308), phospho-Akt (Ser-473), phospho-p44/42 mitogen-activated protein kinase (Thr-202/Tyr-204), phospho-S6 ribosomal protein (Ser-235/236), and total eIF4E.
Figure 7
Figure 7
vMYXV-M64R is highly attenuated in a mouse intranasal infection model while vMYXV-M62R is virulent. (A). Groups of 5 Balb/c mice were infected intranasally with 105 PFU of indicated viruses or mock infected with the same volume of PBS. The mice were then monitored daily for their body weights. For each group, the average of the percentage of body weight change and standard deviation are shown. (B). At day 7 post infection, mice were euthanized and their lungs and spleen were harvested. Approximately 16 mg of spleen and 5 mg of lung from each mouse were homogenized and their viral loads were determined by plaque assay on VERO cells. Error bars represent standard deviation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andrade MA, Ponting CP, Gibson TJ, Bork P. Homology-based method for identification of protein repeats using statistical significance estimates. J Mol Biol. 2000;298(3):521–37. - PubMed
    1. Barrett JW, Shun Chang C, Wang G, Werden SJ, Shao Z, Barrett C, Gao X, Belsito TA, Villenevue D, McFadden G. Myxoma virus M063R is a host range gene essential for virus replication in rabbit cells. Virology. 2007;361(1):123–32. - PubMed
    1. Bradley RR, Terajima M. Vaccinia virus K1L protein mediates host-range function in RK-13 cells via ankyrin repeat and may interact with a cellular GTPase-activating protein. Virus Res. 2005;114(1–2):104–12. - PubMed
    1. Buller RM, Burnett J, Chen W, Kreider J. Replication of molluscum contagiosum virus. Virology. 1995;213(2):655–9. - PubMed
    1. Buller RM, Smith GL, Cremer K, Notkins AL, Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature. 1985;317(6040):813–5. - PubMed

Publication types

Substances